Bioprobes and methods of use thereof

ABSTRACT

Disclosed are biomolecule based bioprobes that exhibit improved water solubility and mono layer-forming properties with substantially little or no aggregation that can appreciably interfere with binding of the bioprobes to a target nucleotide. The bioprobes may be used in conjunction with a suitable reporter system to detect very small quantities of biological markers. The bio-probes comprise a nucleobase sequence capable of hybridizing to a target nucleotide; and at least one charged functional group attached to said nucleobase sequence. Also disclosed are biosensors, and sensing devices that comprise the bin-probe. Further disclosed are suitable electrochemical reporter systems for use with the bioprobes. Methods of use of these devices and probes, including for the detection of target biomarkers, including biomarkers for cancer cells or pathogens, are also included.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/983,934, which is a United States National Stage Application filingunder 35 U.S.C. § 371 of International Application No.PCT/US2012/024015, filed on Feb. 6, 2012, which claims the benefit ofpriority of U.S. Provisional Patent Application Ser. No. 61/440,336,filed Feb. 7, 2011, now expired, both of which are hereby incorporatedby reference herein in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 4, 2016, isnamed 109904-0006-302_SL.txt and is 4,827 bytes in size.

TECHNICAL FIELD

The present disclosure relates generally to biosensors, biosensorarrays, probes for biosensors and a method for detecting biomarkersusing a biosensor. The disclosure also relates to biomolecule basedbioprobes that have reduced or are free of aggregation. Thebiomolecule-based bioprobes may further be used in conjunction with anelectrochemical reporter system to detect small quantities of biologicalmarkers.

BACKGROUND OF THE INVENTION

Detection and analysis of low concentrations of analytes in variousbiologic and organic environments is becoming increasingly important.For a sensing approach to be useful not only for diagnosis, but also tomonitor low levels of residual disease, the limits of detection mustpresently reach low levels, for example femtomolar levels, to achievethe detection of scarce analytes in clinical samples with an acceptablylow level of false negatives. High levels of specificity are required toensure low levels of false positives. From a practical perspective,equally important is a streamlined approach to sample workup, since theneed for extensive sample processing can overwhelm the benefits of asensor's innate high sensitivity and specificity.

Qualitative analysis is generally limited to the higher concentrationlevels, whereas quantitative analysis usually requires labeling with aradioisotope or fluorescent reagent. Such procedures are generally timeconsuming and inconvenient. Recent advances in developing bioelectronicbiological marker analysis systems open up new opportunities formolecular diagnostics and have attracted substantial research efforts(Boon, E. M., et al., Nat. Biotechnol., 18, 1096, 2000; Rodriguez, M. &Bard, A., J. Anal. Chem., 62, 1658, 1990). Optical (Jordan, C. E., etal., Anal. Chem., 69, 4939, 1997; Fotin, A. V., et al., Nucleic AcidsRes., 26, 1515, 1998), electrochemical (Kelley, S. O., et al. Bioconjug.Chem., 8, 31, 1997; Kelly, S. O., et al., Nucleic Acids Res., 27, 4830,1999), and microgravimetric and quartz-crystal microbalance (Bardea, A.,30 et al., Chem. Commun., 839, 1998, Wang, J., Nucleic Acids Res., 28,3011, 2000), transduction methods have been reported for the detectionof DNA hybridization events.

One of the objectives disclosed hereinis to provide biomolecule basedbioprobes that are free of aggregation. Another object is the use of thedisclosed bioprobes useful to detect small quantities of biologicalmarkers. Another objective is to provide biosensors that comprise thebioprobes described herein. An additional objective is to providebiosensors that comprise the bioprobes described herein, immobilized ona suitable substrate, and a suitable reporter system attached thereto.

SUMMARY OF THE INVENTION

Disclosed are methods and systems for the detection and manipulation ofbiomolecules using biomolecule based bioprobes described herein.

This disclosure relates to a new class of bioprobe molecules based onamino acid/nucleic acid chimeras that increase sensitivity andselectivity, and overcome limitations that may arise when using probemolecules, including poor solubility, aggregation, and poor monolayerquality. Also provided are biosensors, comprising the bioprobesdescribed herein. Disclosed herein are biosensors that require only asingle, simple cell lysis step prior to analysis. In some embodiments,the samples tested using the biosensors disclosed herein aresubstantially unpurified.

In one embodiment, disclosed herein are bioprobes that comprise anucleobase sequence capable of hybridizing to a target nucleotide; andat least one charged functional group comprising an anionic functionalgroup, a cationic functional group and/or a charged amino acid attachedto the nucleobase sequence. In certain embodiments, the nucleobasesequence is a nucleic acid sequence such as ribonucleic acid (RNA),deoxyribonucleic acid (DNA) or analog 20 thereof, including, forexample, a morpholino nucleic acid, a methyl phosphonate nucleic acidand a peptide nucleic acid (PNA), which contains a backbone comprised ofN-(2-aminoethyl)-glycine units linked by peptides rather thandeoxyribose or ribose, peptide nucleic acids, locked nucleic acids, orphosphorodiamidate morpholino oligomers. Under appropriate conditions,the probe can hybridize to a complementary nucleic acid to provide anindication of the presence of the complementary nucleic acid in thesample.

In certain embodiments, the anionic functional group is a carboxylate, asulfate or a sulfonate. In certain embodiments, the cationic functionalgroup is an amine or guanadinum group. In some embodiments, the chargedamino acid is an L-amino acid comprising a net positive charge such asLysine (Lys, K), Omithine (Om, O), Diamino-butyric acid (Dab),Diamino-propionic acid (Dap), and Arginine (Arg, R). In someembodiments, the charged amino acid is an L-amino acid or a D-amino acidcomprising a net negative charge such as Aspartic acid (Asp, D),Glutamic acid (Glu, E), or Aminoadipic acid (Aad),4-phosphonomethyl-L-phenylalanine, 4-phosphonomethyl-D-phenylalanine.L-carboxyglutamic acid, D-carboxyglutamic acid, 5-Amino Salicylic Acidor any other charged amino acid from the group shown in Table 2.

In certain embodiments, the probe also comprises a peptide or a proteinthat is able to bind to or otherwise interact with a biomarker target(e.g. receptor or ligand) to provide an 5 indication of the presence ofthe ligand or receptor in the sample. The probe may include a functionalgroup (e.g., thiol, dithiol, amine, carboxylic acid) that facilitatesbinding with an electrode. Probes may also contain other features, suchas longitudinal spacers, double-stranded and/or single-stranded regions,polyT linkers, double stranded duplexes as rigid linkers and PEGspacers. Bioprobes can be immobilized on resins, nanoparticles,nanocrystals, or microparticles.

In some embodiments, the bioprobe is associated with an electrode. Incertain embodiments, the electrode is a microelectrode. In someembodiments, the microelectrode is a nanostructured microelectrode(NME). In further embodiments, the electrodes are present as a pluralityof electrodes arrayed on a substrate.

In certain embodiments the bioprobe associated electrodes may beprepared on a biosensing device, such as a chip-based format, such thata series of electrodes may be made on a single chip to enablemultiplexed experiments.

In certain embodiments are provided biosensing devices, such asintegrated circuits, comprising, for example, a substrate; anelectrically conductive lead on the substrate; an insulating orpassivation layer covering the lead, the insulating layer having anaperture exposing a portion of the lead; and an electrode in electricalcommunication with the exposed portion of the lead, the electrode beingadapted to generate a charge in response to a biomolecular stimulus suchas hybridization or interaction.

In some embodiments are methods for carrying out a biosensing processusing electrodes containing a bioprobe incorporated into a device;biasing the microelectrode relative to a reference electrode; measuringa reference charge or reference current flow between the microelectrodeand the reference electrode; exposing the electrode containing thebioprobe to a biomolecular stimulus (e.g., hybridization with acomplementary nucleic acid or binding with a binding partner present ina biological sample); measuring a charge or current flow generated atthe microelectrode in response to the biomolecular stimulus; anddetermining the amount of biomolecular stimulus present by comparing themeasured charge or measured current flow against the reference charge orreference current flow.

In certain embodiments are methods of detecting a biomolecule or abiomarker, using the bioprobes and biosensing devices described herein.The method involves contacting a purified or unpurified sample with abiosensing device that comprises a bio-probe comprising a nucleobasesequence capable of hybridizing to the target biomarker of interest, andat least one charged functional group attached to said nucleobasesequence; hybridizing the bio-probe to the target biomarker; anddetecting the hybridization as being indicative of presence of thebiomarker of interest in the sample. In certain embodiments, thisdetection is performed by means of a redox-active reporter. In certainembodiments, this method is used to detect the presence of targetbiomolecules in solutions, such as biological fluids obtained from atest subject.

In some embodiments, provided are methods of identification of aspecific biomolecule (e.g., nucleic acids) within a population ofheterogeneous biomolecules. The method comprises contacting a bio-probecomprising a nucleobase sequence capable of hybridizing to the targetbiomolecule of interest, and at least one charged functional groupattached to said nucleobase sequence; hybridizing the bio-probe to thetarget biomolecule; and detecting the hybridization as being indicativeof presence of the biomolecule of interest.

In certain embodiments are methods of diagnosing a disease or conditionin a subject, using the bioprobes and biosensing devices describedsupra. In certain embodiments, the disease is a cancer. In certainembodiments is a method of detecting the presence of cancer cells in abiological sample, comprising: contacting the sample with a bio-probecomprising a nucleobase sequence capable of hybridizing to a targetbiomarker, and at least one charged functional group attached to saidnucleobase sequence; hybridizing the bio-probe to the target genesequence; and detecting the hybridization as being indicative of thepresence of cancer cells in the sample.

In some embodiments, the cancer being diagnosed or detected is selectedfrom the group consisting of breast cancer, skin cancer, bone cancer,prostate cancer, liver cancer, lung cancer, brain cancer, cancer of thelarynx, gall bladder, pancreas, rectum, parathyroid, thyroid, adrenal,neural tissue, head and neck, colon, stomach, bronchi, kidneys, basalcell carcinoma, squamous cell carcinoma of both ulcerating and papillarytype, metastatic skin carcinoma, melanoma, osteosarcoma, Ewing'ssarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-celllung tumor, gallstones, islet cell tumor, primary brain tumor, acute andchronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma,hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neurons,intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoidhabitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomatertumor, cervical dysplasia and in situ carcinoma, neuroblastoma,retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skinlesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenicand other sarcoma, malignant hypercalcemia, renal cell tumor,polycythermia vera, adenocarcinoma, glioblastoma multiforma, leukemias,lymphomas, malignant melanomas, and epidermoid carcinomas. In otherembodiments, the cancer being treated is pancreatic cancer, livercancer, breast cancer, osteosarcoma, lung cancer, soft tissue sarcoma,cancer of the larynx, melanoma, ovarian cancer, brain cancer, Ewing'ssarcoma or colon cancer.

In yet another embodiment, a method for conducting a business, includingproviding biosensors disclosed herein to a physician or health careprovider, is provided.

In other embodiments, the sample is obtained from a subject which is amammal, preferably a human. In some embodiments, the sample may includeblood, urine, semen, milk, sputum, mucus, a buccal swab, a vaginal swab,a rectal swab, an aspirate, a needle biopsy, a section of tissueobtained for example by surgery or autopsy, plasma, serum, spinal fluid,lymph fluid, the external secretions of the skin, respiratory,intestinal, and genitourinary tracts, tears, saliva, tumors or organs.

These and other aspects, embodiments, objects and features disclosedherein will be more fully appreciated when the following detaileddescription is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E depict chip-based biosensors.

FIG. 1A shows the layout of the chip wherein a gold pattern is depositedon the chip surface with 8 external contacts that are extended to narrowleads of a terminal width of 5 microns. A passivating layer of silicondioxide is applied to the chip, and then 5 micron apertures are openedat the end of each lead to provide a microelectrode template.

FIG. 1B is a scanning electron micrograph of a 100 micron sensor formedusing gold electrodeposition on the surface of the chip.

FIG. 1C shows sequence of steps used for nucleic acids analysis. Thesensors are first functionalized with probe molecules, and thenhybridized with a target-containing solution.

FIG. 1D shows Ru(III)/Fe(III) reporter groups then permits hybridizedmaterial to be detected.

FIG. 1E depicts the overall flow of analysis trial.

FIGS. 2A-2D show the target and the bioprobes.

FIG. 2A depicts the junction region between the bcr gene and the ablgene within the mRNA expressed that generates the bcr-abl kinase.

FIG. 2B shows structures of DNA, PNA, and ANA (amino-acid/nucleic acidschimera) bioprobes.

FIG. 2C shows testing of DNA, PNA and ANA probes for hybridization ofmRNA isolated from the K562 cell line that carries the bcr-abl genefusion

FIG. 2D shows different placement options for charged amino acids inconjugation with a 20 nucleobase probe sequence to generate bioprobesdescribed here.

FIG. 3 shows Sensitivity of biosensors modified with an ANA bioprobetowards the mRNA from the K.562 cell line which carries the bcr-abl genefusion.

FIGS. 4A-4E depict performance of ANA-modified sensors when challengedwith crude cell lysates.

FIG. 4A shows K562 lysates wherein signals obtained before and afterincubation of probe-modified microelectrodes with lysates of K562 cellswere compared by monitoring the limiting reductive current in aRu(III)/Fe(III) electrocatalysis solution. Total volume of samples was30 microliters.

FIGS. 4B and 4C are representative differential pulse voltammogramsshowing the change in signal when lysates containing 10 and 1000 cellswere introduced. Dotted lines represent the signal collected beforehybridization, and the solid lines are signals collected afterhybridization.

FIG. 4D shows detection of the bcr-abl gene fusion in CML patientleukocytes. Lysates were generated from 10-1000 cells, and signalchanges monitored after 30 minutes. The presence of the bcr-abl fusionwas confirmed using PCR (inset). Primers specific to each gene were usedto analyze a fragment of the fusion, and used with K562 cells (leftband) and patient cells (right band).

FIG. 4E shows detection of the bcr-abl gene fusion in whole blood.

FIG. 5 shows optimization of biosensor deposition conditions: variationof electroplating potential to increase sensor footprint. 0 mV wasidentified as the optimal potential to use in order to generate 100micron sensors.

FIG. 6 shows representative differential pulse voltammograms obtainedwith three 25 different probe types before (dotted) and after (solid)solution of K562 mRNA with a concentration of 1 ng/ul.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, disclosed herein are bioprobes that comprise anucleobase sequence capable of hybridizing to a target nucleotide; andat least one charged functional group comprising at least one anionicfunctional group, cationic functional group and/or a charged amino acidattached to the nucleobase sequence. In some embodiments, the nucleobasesequence is a nucleic acid sequence such as ribonucleic acid (RNA),deoxyribonucleic acid (DNA) or analog thereof, including, for example, apeptide nucleic acid (PNA), which contains a backbone comprised ofN-(2-aminoethyl)-glycine units linked by peptides rather thandeoxyribose or ribose, peptide nucleic acids, locked nucleic acids, orphosphorodiamidate morpholino oligomers. Under appropriate conditions,the probe can hybridize to a complementary nucleic acid to provide anindication of the presence of the nucleic acid in the sample.

In certain embodiments, the anionic functional group is a carboxylate,sulfate or sulfonate. In certain embodiments, the cationic functionalgroup is an amine or guanadinum group. In certain embodiments, thecharged amino acid is a chiral amino acid, which is an L-amino acid or aD-amino acid. In certain embodiments, the amino acid comprises a netpositive charge such as Lysine (Lys, K), Omithine (Om, O),Diamino-butyric acid (Dab), Diamino-propionic acid (Dap), and Arginine(Arg, R). In certain embodiments, the charged amino acid 10 comprises anet negative charge such as Aspartic acid (Asp, D), Glutamic acid (Glu,E), Aminoadipic acid (Aad), 4-phosphonomethyl-L-phenylalanine,4-phosphonomethyl-D-phenylalanine, L-carboxyglutamic acid,D-carboxyglutamic acid, 5-Amino Salicylic Acid or any other chargedamino acid selected from the group shown in Table 2.

In one embodiment, disclosed herein is a bio-probe, comprising:

-   -   a nucleobase sequence capable of hybridizing to a target        nucleotide;    -   at least one charged functional group attached to said        nucleobase sequence, wherein said charged functional group        comprises a cationic functional group, an anionic functional        group, a charged amino acid, or a combination thereof; and    -   wherein attachment of said charged functional group to said        nucleobase results in lesser aggregation of a plurality of        bio-probes, as compared to bio-probes not comprising a charged        functional group attached to said nucleobase.

In one embodiment, the bioprobes do not aggregate with a plurality ofbio-probes. In another embodiment, the nucleobase sequence is a DNA,RNA, PNA, morpholino nucleic acid, or methyl phosphonate nucleic acidoligomer. In yet another embodiment, the nucleobase sequence is a PNA.In another embodiment, the anionic functional group is a carboxylate, asulfate or a sulfonate. In still another embodiment, the cationicfunctional group is an amine or a guanadinum. In other embodiments, theamino acid is Asp, Glu, Aad, Ser, Lys, Om, Dab, Dap, Arg,4-phosphonomethyl-L-phenylalanine, 4-phosphonomethyl-D-phenylalanine,L-carboxyglutamic acid, D-carboxyglutamic acid, 5-Amino Salicylic Acid,or an amino acid shown 30 in Table 2.

In some embodiments, the bio-probe comprises between about 10 and about40 nucleobases. In other embodiments, the bio-probe comprises betweenabout 1 and about 20 charged functional groups. In still otherembodiments, the charged functional groups are arranged as shown inTable 3. In some embodiments, the bio-probes are immobilized to asurface, wherein the surface may be nitrocellulose, nylon membrane,glass plate, or a polyvinyldifluoride surface. In some embodiments, thesurface may be a multi-well plate, a resin, a nanoparticle, ananocrystal, or a microparticle. In other embodiments, the bioprobe isimmobilized to an electrode. In still other embodiments, the bio-probefurther comprises a redox 5 label, wherein the bioprobe is capable ofbinding to a redox reporter when hybridized to a nucleic acid sequence.In some embodiments, aggregation of the bioprobe, as compared tobioprobes lacking the charged functional group, is decreased by at least10%, at least 20%, at least at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90% or at least 100%. Inyet other embodiments, the aggregation is decreased by between about 30%and about 90%, or by between about 50% and about 80%.

In some embodiments, a method for detecting a biomarker of interest isprovided, the method comprising:

-   -   contacting the sample with a bio-probe comprising a nucleobase        sequence capable of hybridizing to the biomarker of interest,        and at least one charged functional group attached to said        nucleobase sequence, wherein attachment of said charged        functional group to said nucleobase results in lesser        aggregation of a plurality of bio-probes; hybridizing the        bio-probe to the biomarker; and detecting the hybridization as        being indicative of presence of the biomarker of interest in the        sample.

In some embodiments, the charged functional group is a cationicfunctional group, an anionic functional group, a charged amino acid or acombination thereof. In some embodiments, the anionic functional groupis a carboxylate, a sulfate or a sulfonate. In other embodiments, thecationic functional group is an amine or a guanadinum. In yet otherembodiments, the amino acid is Asp, Glu, Aad, Ser, Lys, Orn, Dab, Dap,Arg, 4-phosphonomethyl-L-phenylalanine,4-phosphonomethyl-D-phenylalanine, L-carboxyglutamic acid,D-carboxyglutamic acid, 5-Amino Salicylic Acid, or an amino acid shownin Table 2.

In still other embodiments, an increase in signal of at least 10%, atleast 20%, at least 30%, at least 40%, at least 50% at least 60%, atleast 70%, at least 80%, at least 90%, at least 100%, at least 125% orat least 150% over a control signal is detectable upon hybridization. In30 other embodiments, an increase in signal of at least 20%-100% over acontrol signal is detectable upon hybridization. In still otherembodiments, an increase in signal of at least 25%-50% over a controlsignal is detectable upon hybridization. In some embodiments, thebioprobes are immobilized onto a solid surface. In other embodiments, aplurality of bioprobes are immobilized onto distinct locations on thesolid surface. In some embodiments, detection is performed by observinga reporter signal, and wherein a change in reporter signal onhybridization of the bio-probe with the biomaker, as compared to saidreporter signal in the absence of hybridization of said bioprobe, isindicative of presence of said biomarker in the sample. In someembodiments, the reporter signal decreases on hybridization of thebio-probe with the biomarker. In some embodiments, the reporter signaldecreases by about 10% to about 30% on hybridization of the bio-probewith the biomarker. In some embodiments, the reporter signal decreasesby about 20% to about 50% on hybridization of bio-probe with thebiomarker. In some embodiments, the reporter signal decreases by about50% to about 100% on hybridization of bio-probe with the biomarker.

In some embodiments, the detection is by means of a fluorescent reportergroup. In other embodiments, the detection is by means of a FRET system.In other embodiments, the detection is by means of a dye. In still otherembodiments, the detection is by means of a redox reporter system,wherein the redox reporter system may be water-soluble. In someembodiments, the redox reporter group may comprise a metal, wherein themetal may be one of copper (Cu), cobalt 15 (Co), palladium (Pd), iron(Fe), ruthenium (Ru), rhodium (Rh), osmium (Os), rhenium (Re), platinum(Pt), scandium (Sc), titanium (Ti), vanadium (V), chromium (Cr),manganese (Mn), nickel (Ni), molybdenum (Mo), technetium (Tc), tungsten(W), and iridium (Ir). In some embodiments, the nucleobase sequence is aDNA oligomer, an RNA oligomer or a PNA oligomer. In some embodiments,the aggregation of the bioprobe, as compared to bioprobes lacking thecharged functional group, is decreased by at least 10%, at least 20%, atleast at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90% or at least 100%. In yet otherembodiments, the aggregation is decreased by between 30%-90%, or bybetween 50%-80%.

In one embodiment, the target biomarker is DNA, RNA or a protein. Insome embodiments, the sample may be a biological fluid, wherein thebiological fluid may be selected from aqueous humour, vitreous humour,blood serum, breast milk, cerebrospinal fluid, cerumen, endolymph andperilymph, sastric juice, mucus (including nasal drainage and phlegm),peritoneal fluid, pleural fluid, saliva, sebum, semen, sweat, tears,vaginal secretion, vomit and urine.

A method for detecting the presence of a biomarker in an individual isalso provided herein, the method comprising:

-   -   obtaining a biological fluid sample from the individual;    -   contacting the sample with a bio-probe comprising a nucleobase        sequence capable of hybridizing to said biomarker, and at least        one charged functional group attached to said nucleobase        sequence; wherein attachment of said charged functional group to        said nucleobase results in lesser aggregation of a plurality of        bio-probes; hybridizing the bio-probe to the biomarker gene; and        detecting the hybridization as being indicative of presence of        the biomarker in the individual.

Also provided herein are methods of detecting the presence of cancercells in a biological sample, the method comprising:

-   -   contacting the sample with a bio-probe comprising a nucleobase        sequence capable of hybridizing to a target biomarker, and at        least one charged functional group attached to said nucleobase        sequence; wherein attachment of said charged functional group to        said nucleobase results in lesser aggregation of a plurality of        bio-probes;    -   hybridizing the bio-probe to the target gene sequence; and        detecting the hybridization as being indicative of presence of        cancer cells in the sample.

In some embodiments, the cancer cells detected are breast cancer, skincancer, bone cancer, prostate cancer, liver cancer, lung cancer, braincancer, cancer of the larynx, gall bladder, pancreas, rectum,parathyroid, thyroid, adrenal, neural tissue, head and neck, colon,stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinomaof both ulcerating and papillary type, metastatic skin carcinoma,melanoma, osteosarcoma, Ewing's sarcoma, veticulum cell sarcoma,myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet celltumor, primary brain tumor, acute and chronic lymphocytic andgranulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullarycarcinoma, pheochromocytoma, mucosal neurons, intestinalganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitustumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor,cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma,soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosisfungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and othersarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera,adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignantmelanomas or epidermoid carcinomas.

In yet other embodiments, also provided are methods of detecting thepresence of cancer cells in an individual, comprising:

-   -   obtaining a biological fluid sample from the individual;    -   contacting the sample with a bio-probe comprising a nucleobase        sequence capable of hybridizing to a target biomarker, and at        least one charged functional group attached to said nucleobase        sequence, wherein attachment of said charged functional group to        said nucleobase results in lesser aggregation of a plurality of        bio-probes; hybridizing the bio-probe to the target gene        sequence; and    -   detecting the hybridization as being indicative of presence of        cancer cells in the sample.

In still other embodiments, provided are biosensors comprising:

-   -   an electrode; and    -   a bio-probe comprising a nucleobase sequence capable of        hybridizing to a target biomarker, and at least one charged        functional group attached to said nucleobase sequence, wherein        attachment of said charged functional group to said nucleobase        results in lesser aggregation of a plurality of bio-probes.

In yet other embodiments, provided are biosensing devices comprising:

-   -   a bio-probe comprising a nucleobase sequence capable of        hybridizing to a target biomarker, and at least one charged        functional group attached to said nucleobase sequence, wherein        attachment of said charged functional group to said nucleobase        results in lesser aggregation of a plurality of bio-probes; and        at least one redox active reporter.

In certain embodiments, the probe also comprises a peptide or a proteinthat is able to bind to or otherwise interact with a biomarker target(e.g. receptor or ligand) to provide an indication of the presence ofthe ligand or receptor in the sample. The probe may include a functionalgroup (e.g., thiol, dithiol, amine, carboxylic acid) that facilitatesbinding with an electrode. Probes may also contain other features, suchas longitudinal spacers, double-stranded and/or single-stranded regions,polyT linkers, double stranded duplexes as rigid linkers and PEGspacers.

In some embodiments, the bioprobe is bound or otherwise associated witha substrate. In some embodiments, the substrate may be a solid surface.In other embodiments, the substrate may be nitrocellulose, a nylonmembrane, a glass plate, or polyvinyldifluoride (“PVDF”), a multiwellplate or other substrate, such as the tip of a light guide, opticalfiber, conducting material or biosensor device. Further examples ofsubstrates include materials that are comprised of a semiconductormaterial, such as silicon, silica, quartz, germanium, gallium arsenide,silicon carbide and indium compounds (e.g., indium arsenide, indium,antimonide and indium phosphide), selenium sulfide, ceramic, glass,plastic, polycarbonate or other polymer or combinations of any of theabove. Substrates may optionally include a passivation layer, which iscomprised of a material, which offers high resistance and maintains asmall active surface area. Examples of appropriate materials include:silicon dioxide, silicon nitride, nitrogen doped silicon oxide (SiOxNy)or paralyene. In some embodiments, a linker or other spacer may be usedto bind or otherwise associate the bioprobes disclosed herein with thesubstrate.

In some embodiments, the bioprobe is associated with an electrode.Electrodes may be comprised of a noble metal, (e.g., gold, platinum,palladium, silver, osmium, indium, rhodium, ruthenium); alloys of noblemetals (e.g., gold-palladium, silver-platinum, etc.); conductingpolymers (e.g., polypyrole (PPY)); non-noble metals (e.g., copper,nickel, aluminum, tin, titanium, indium, tungsten, platinum); metaloxides (e.g., zinc oxide, tin oxide, nickel oxide, indium tin oxide,titanium oxide, nitrogen-doped titanium oxide (TiOxNy); metal suicides(nickel suicide, platinum suicide); metal nitrides (titanium nitride(TiN), tungsten nitride (WN) or tantalum nitride (TaN)), carbon(nanotubes, fibers, graphene and amorphous) or combinations of any ofthe above.

In certain embodiments, the electrode is a microelectrode. Exemplarymicroelectrodes have a height in the range of about 0.5 to about 100microns (μm), for example in the range of about 5 to about 20 microns(e.g., 10 microns); a diameter in the range of about 0.1 to about 500microns, for example in the range of about 1 to about 100 microns, orfor example in the range of about 1 to about 50 microns, or for examplein the range of about 1 to about 1 microns. Microelectrodes can be anyof a variety of shapes, including hemispherical, irregular (e.g.,spiky), cyclical (wire-like) or fractal (e.g., dendritic). The surfaceof a microelectrode may be further coated or functionalized with amaterial which maintains the electrode's high conductivity, butfacilitates binding with a probe.

In other embodiments, the electrode is a nanostructured microelectrode(NME). NMEs are electrodes, which are nanotextured and thus have anincreased surface area. NMEs of the above-described materials are highlyconductive and form strong bonds with the bioprobes. Exemplary NMEs havea height in the range of about 0.5 to about 100 microns (μm), forexample in the range of about 5 to about 20 microns (e.g., 10 microns);a diameter in the range of about 1 to about 100 microns, for example inthe range of about 1 to about 50 microns, or for example in 25 the rangeof about 1 to about 10 microns; and have nanoscale morphology (e.g., arenanostructured on a length scale of about 1 to about 300 nanometers andmore preferably in the range of about 10 to about 20 nanometers). NMEscan be any of a variety of shapes, including hemispherical, irregular(e.g., spiky), cyclical (wire-like) or fractal (e.g., dendritic). Thesurface of an NME may be further coated or functionalized with amaterial, which maintains the electrode's high conductivity, butfacilitates binding with a probe. For example, nitrogen containing NMEs(e.g., TiN, WN or TaN) can bind with an amine functional group of theprobe. Similarly, silicon/silica chemistry as part of the NME can bindwith a silane or siloxane group on the probe.

In a further embodiment a plurality of electrodes are arrayed on asubstrate. Exemplary substrates are comprised of a semiconductormaterial, such as silicon, silica, quartz, germanium, gallium arsenide,silicon carbide and indium compounds (e.g., indium arsenide, indium,antimonide and indium phosphide), selenium sulfide, ceramic, glass,plastic, polycarbonate or other polymer or combinations of any of theabove. Substrates may optionally include a passivation layer, which iscomprised of a material, which offers high resistance and maintains asmall active surface area. Examples of appropriate materials include:silicon dioxide, silicon nitride, nitrogen doped silicon oxide (SiOxNy)or paralyene. In certain embodiments, the plurality of electrodes arearrayed on the substrate, comprise bioprobes in conjunction withmonolayer spacers, which minimize probe density, thereby maximizingcomplexation efficiency. Exemplary monolayer spacers have an affinity tometal and can be comprised, for example, of a thiol alcohol, such asmercaptohexanol, alkanethiols, cysteine, cystamine, thiol-amines,aromatic thiols (e.g., benzene thiol, dithiol), phosphonic acids orphosphinic acids.

The present disclosures may comprise in its embodiments any addressablearray technology known in the art. One embodiment of polynucleotidearrays has been generally described in U.S. Pat. No. 5,143,854; PCTpublications WO 90/15070 and 92/10092. These arrays may generally beproduced using mechanical synthesis methods or light directed synthesismethods, which incorporate a combination of photolithographic methodsand solid phase oligonucleotide synthesis. (Fodor et al, Science,251:767-777, (1991)). The immobilization of arrays of oligonucleotideson solid supports has been rendered possible by the development of atechnology generally identified as “Very Large Scale Immobilized PolymerSynthesis” in which, typically, probes are immobilized in a high densityarray on a solid surface of a chip. Examples of such technologies areprovided in U.S. Pat. Nos. 5,143,854 and 5,412,087 and in PCTPublications WO 90/15070. WO 92/10092 and WO 95/11995, which describemethods for forming oligonucleotide arrays through techniques such aslight-directed synthesis techniques. In designing strategies aimed atproviding arrays of nucleotides immobilized on solid supports, furtherpresentation strategies to order and display the oligonucleotide arrayson the chips in an attempt to maximize hybridization patterns andsequence information are disclosed in PCT Publications WO 94/12305, WO94/11530, WO 97/29212 and WO 97/31256.

In certain embodiments are methods of detecting a biomolecule or abiomarker, using the bioprobes and biosensing devices described herein.The method involves contacting a purified or unpurified sample with abiosensing device that comprises a bio-probe comprising a nucleobasesequence capable of hybridizing to the target biomarker of interest, andat least one charged functional group, including a cationic functionalgroup, a anionic functional group or a charged amino acid, attached tosaid nucleobase sequence; hybridizing the bio-probe to the targetbiomarker; and detecting the hybridization as being indicative ofpresence of the biomarker of interest in the sample. In certainembodiments, this detection is performed by means of a redox-activereporter. In certain embodiments, this method is used to detect thepresence of target biomolecules in solutions, such as biological fluidsobtained from a test subject. These biological fluids include, but arenot limited to aqueous humour, vitreous humour, blood serum, breastmilk, cerebrospinal fluid, cerumen (earwax), endolymph and perilymph,sastric juice, mucus (including nasal drainage and phlegm), peritonealfluid, pleural fluid, saliva, sebum (skin oil), semen, sweat, tears,vaginal secretion, vomit and urine.

In some embodiments, is provided a method of identification of aspecific biomolecule (e.g., nucleic acids) within a population ofheterogeneous biomolecules. The method comprises contacting a bio-probecomprising a nucleobase sequence capable of hybridizing to the targetbiomolecule of interest, and at least one charged amino acid attached tosaid nucleobase sequence; hybridizing the bio-probe to the targetbiomolecule; and detecting the hybridization as being indicative ofpresence of the biomolecule of interest.

In certain embodiments are methods of diagnosing a disease or conditionin a subject, using the bioprobes and biosensing devices describedsupra. In certain embodiments, the disease is a cancer. In certainembodiments is a method of detecting the presence of cancer cells in abiological sample, comprising: contacting the sample with a bio-probecomprising a nucleobase sequence capable of hybridizing to a targetbiomarker, and at least one charged amino acid attached to saidnucleobase sequence; hybridizing the bio-probe to the target genesequence; and detecting the hybridization as being indicative ofpresence of cancer cells in the sample.

In some embodiments, the cancer being diagnosed or detected is selectedfrom the group consisting of breast cancer, skin cancer, bone cancer,prostate cancer, liver cancer, lung cancer, brain cancer, cancer of thelarynx, gall bladder, pancreas, rectum, parathyroid, thyroid, adrenal,neural tissue, head and neck, colon, stomach-bronchi, kidneys, basalcell carcinoma, squamous cell carcinoma of both ulcerating and papillarytype, metastatic skin carcinoma, melanoma, osteosarcoma, Ewing'ssarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-celllung tumor, gallstones, islet cell tumor, primary brain tumor, acute andchronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma,hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms,intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoidhabitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomatertumor, cervical dysplasia and in situ carcinoma, neuroblastoma,retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skinlesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenicand other sarcoma, malignant hypercalcemia, renal cell tumor,polycythermia vera, adenocarcinoma, glioblastoma multiforma, leukemias,lymphomas, malignant melanomas, and epidermoid carcinomas. In otherembodiments, the cancer is pancreatic cancer, liver cancer, breastcancer, osteosarcoma, lung cancer, soft tissue sarcoma, cancer of thelarynx, melanoma, ovarian cancer, brain cancer, Ewing's sarcoma or coloncancer.

In certain embodiments are methods of detecting a pathogen using thebioprobes and biosensing devices described herein. In certainembodiments, the pathogen is a bacteria, a virus, a fungus or aparasite. In certain embodiments is a method of detecting the presenceof a pathogen in a biological sample, comprising: contacting the samplewith a bio-probe comprising a nucleobase sequence capable of hybridizingto a target biomarker, and at least one charged amino acid attached tosaid nucleobase sequence, hybridizing the bio-probe to the target genesequence; and detecting the hybridization as being indicative ofpresence of a pathogen, including a bacteria, a virus, a fungus, or aparasite, in the sample.

The embodiments of the multiplexing methods disclosed herein can be usedto screen a single individual against a battery of the bio-probesdescribed herein. Accordingly, different types of bio-probes may belabeled with different reporters so that the presence of multiplebioprobes bound to target biomolecules from a biological sample from theindividual can be accomplished. Alternatively, the different bioprobesmay be attached to distinct positions on a solid substrate, such thatdetection of bioprobes bound to specific target biomolecules form abiological sample from the individual can be discerned. A biologicalsample from the individual to be screened is obtained and prepared andthe biomolecules to be probed are disposed on a support. The variousbioprobes are contacted with the biomolecules, the unbound andnonspecifically bound probes are removed by washing, and the signals aredetected and resolved.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the technology belongs. All patents, patent applications,published applications and publications, Genbank sequences, websites andother published materials referred to throughout the entire disclosureherein, unless noted otherwise, are incorporated by reference in theirentirety. In the event that there is a plurality of definitions forterms herein, those in this section prevail. Where reference is made toa URL or other such identifier or address, it is understood that suchidentifiers can change and particular information on the internet cancome and go, but equivalent information can be found by searching theinternet. Reference thereto evidences the availability and publicdissemination of such information.

As used herein, the term “nucleobase” as used herein, is intended to bysynonymous with “nucleic acid base or mimetic thereof.” In general, anucleobase is any substructure that contains one or more atoms or groupsof atoms capable of hydrogen bonding to a base of a nucleic acid.

As used herein, “unmodified” or “natural” nucleobases include the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, and 10alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine 15 and3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases,size-expanded bases, and fluorinated bases as defined herein. Furthermodified nucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimidol[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1-H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido(3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer 25 Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15. AntisenseResearch and Applications, pages 289-302, Crooke, S. T, and Lebleu, B.,ed., CRC Press, 1993.

Modified nucleobases include, but are not limited to, universal bases,hydrophobic bases, 30 promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. Certain of these nucleobases areparticularly useful for increasing the binding affinity of theoligomeric compounds disclosed herein. These include 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T, andLebleu. B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121;5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941 each of which is herein incorporated by reference.

The term “nucleobase” also encompasses polymers having additionalsubstituents including, without limitation, protein groups, lipophilicgroups, intercalating agents, diamines, folic acid, cholesterol andadamantane. The term “nucleobase” also encompasses any other nucleobasecontaining polymer, including, without limitation, peptide nucleic acids(PNA), peptide nucleic acids with phosphate groups (PHONA), lockednucleic acids (LNA), morpholino nucleic acids, methyl phosphonatenucleic acids, and oligonucleotides having backbone sections with alkyllinkers or amino linkers.

As used herein, DNA is meant to include all types and sizes of DNAmolecules including cDNA, plasmids and DNA including modifiednucleotides and nucleotide analogs.

As used herein, nucleotides include nucleoside mono-, di-, andtriphosphates. Nucleotides also include modified nucleotides, such as,but are not limited to, phosphorothioate nucleotides and deazapurinenucleotides and other nucleotide analogs.

As used herein, the term “subject” refers to animals, plants, insects,and birds. Included are higher organisms, such as mammals and birds,including humans, primates, rodents, cattle, pigs, rabbits, goats,sheep, mice, rats, guinea pigs, cats, dogs, horses, chicken and others.

As used herein, “selectable or screenable markers” confer anidentifiable change to a cell permitting easy identification of cellscontaining an expression vector. Generally, a selectable marker is onethat confers a property that allows for selection. A positive selectablemarker is one in which the presence of the marker allows for itsselection, while a negative selectable marker is one in which itspresence prevents its selection. An example of a positive selectablemarker is a drug resistance marker.

As used herein, “expression” refers to the process by which nucleic acidis translated into peptides or is transcribed into RNA, which, forexample, can be translated into peptides, polypeptides or proteins. Ifthe nucleic acid is derived from genomic DNA, expression may, if anappropriate eukaryotic host cell or organism is selected, includesplicing of the mRNA. For heterologous nucleic acid to be expressed in ahost cell, it must initially be delivered into the cell and then, oncein the cell, ultimately reside in the nucleus.

The term “host cell” denotes, for example, microorganisms, yeast cells,insect cells, and mammalian cells, that can be, or have been, used asrecipients for multiple constructs for producing a targeted deliveryvector. The term includes the progeny of the original cell which hasbeen transfected. Thus, a “host cell” as used herein generally refers toa cell which has been transfected with an exogenous DNA sequence. It isunderstood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement as the original parent, due to natural, accidental, ordeliberate mutation.

As used herein, “heterologous nucleic acid sequence” is typically DNAthat encodes RNA and proteins that are not normally produced in vivo bythe cell in which it is expressed or that mediates or encodes mediatorsthat alter expression of endogenous DNA by affecting transcription,translation, or other regulatable biochemical processes. A heterologousnucleic acid sequence may also be referred to as foreign DNA. Any DNAthat one of skill in the art would recognize or consider as heterologousor foreign to the cell in which it is expressed is herein encompassed byheterologous DNA. Examples of heterologous DNA include, but are notlimited to, DNA that encodes traceable marker proteins, such as aprotein that confers drug resistance, DNA that encodes therapeuticallyeffective substances, such as anti-cancer agents, enzymes and hormones,and DNA that encodes other types of proteins, such as antibodies.Antibodies that are encoded by heterologous DNA may be secreted orexpressed on the surface of the cell in which the heterologous DNA hasbeen introduced.

The term “moiety” refers to a specific segment or functional group of amolecule. Chemical moieties are often recognized as chemical entitiesembedded in or appended to a molecule.

The term “solid support” refers to supports used for anchoring thebioprobes described herein. The solid support can be a solid surfacesuch as nitrocellulose, a nylon membrane, a glass plate, a resin,nanoparticles, nanocrystals, microparticles, semi-conductor material oran electrode.

The term “linking moiety” refers to any moiety optionally positionedbetween the terminal nucleoside and the solid support or between theterminal nucleoside and another nucleoside, nucleotide, or nucleic acid.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences can be5 described herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the non-transcribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

A DNA “coding sequence” or “coding region” is a double-stranded DNAsequence which is transcribed and translated into a polypeptide in vivowhen placed under the control of appropriate expression controlsequences. The boundaries of the coding sequence (the “open readingframe” or “ORF”) are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. Apolyadenylation signal and 15 transcription termination sequence is,usually, be located 3′ to the coding sequence. The term “non-codingsequence” or “non-coding region” refers to regions of a polynucleotidesequence that are not translated into amino acids (e.g., 5′ and 3′un-translated regions).

The term “reading frame” refers to one of the six possible readingframes, three in each direction, of the double stranded DNA molecule.The reading frame that is used determines which codons are used toencode amino acids within the coding sequence of a DNA molecule.

As used herein, an “antisense” nucleic acid molecule comprises anucleotide sequence which is complementary to a “sense” nucleic acidencoding a protein, e.g., complementary to the coding strand of adouble-stranded cDNA molecule, complementary to an mRNA sequence orcomplementary to the coding strand of a gene. Accordingly, an antisensenucleic acid molecule can hydrogen bond to a sense nucleic acidmolecule.

The term “base pair” or (“bp”): a partnership of adenine (A) withthymine (T), or of cytosine (C) with guanine (G) in a double strandedDNA molecule. In RNA, uracil (U) is substituted for thymine.

As used herein a “codon” refers to the three nucleotides which, whentranscribed and translated, encode a single amino acid residue; or inthe case of UUA, UGA or UAG encode a termination signal. Codons encodingamino acids are well known in the art and are provided for convenienceherein in Table 1.

TABLE 1 Codon Usage Table Codon Amino acid AA Abbr. Codon Amino acid AAAbbr. UUU Phenylalanine Phe F UCU Serine Ser S UUC Phenylalanine Phe FUCC Serine Ser S UUA Leucine Leu L UCA Serine Ser S UUG Leucine Leu LUCG Serine Ser S CUU Leucine Leu L CCU Proline Pro P CUC Leucine Leu LCCC Proline Pro P CUA Leucine Leu L CCA Proline Pro P CUG Leucine Leu LCCG Proline Pro P AUU Isoleucine Ile I ACU Threonine Thr T AUCIsoleucine Ile I ACC Threonine Thr T AUA Isoleucine Ile I ACA ThreonineThr T AUG Methionine Met M ACH Threonine Thr T GUU Valine Val V GCUAlanine Ala A GUC Valine Val V GCC Alanine Ala A GUA Valine Val V GCAAlanine Ala A GUG Valine Val V GCG Alanine Ala A UAU Tyrosine Tyr Y UGUCysteine Cys C UAC Tyrosine Tyr Y UGC Cysteine Cys C UUA Stop UGA StopUAG Stop UGG Tryptophan Trp W CAU Histidine His H CGU Arginine Arg R CACHistidine His H CGC Arginine Arg R CAA Glutamine Gln Q CGA Arginine ArgR CAG Glutamine Gln Q CGG Arginine Arg R AAU Asparagine Asn N AGU SerineSer S AAC Asparagine Asn N AGC Serine Ser S AAA Lysine Lys K AGAArginine Arg R AAG Lysine Lys K AGG Arginine Arg R GAU Aspartate Asp DGGU Glycine Gly G GAC Aspartate Asp D GGC Glycine Gly G GAA GlutamateGlu E GGA Glycine Gly G GAG Glutamate Glu E GGG Glycine Gly G

As used herein, a “wobble position” refers to the third position of acodon. Mutations in a DNA molecule within the wobble position of acodon, in some embodiments, result in silent or conservative mutationsat the amino acid level. For example, there are four codons that encodeGlycine, i.e., GGU, GGC, GGA and GGG, thus mutation of any wobbleposition nucleotide, to any other nucleotide, does not result in achange at the amino acid level of the encoded protein and, therefore, isa silent substitution.

As used herein, a “silent substitution” or “silent mutation” is one inwhich a nucleotide within a codon is modified, but does not result in achange in the amino acid residue encoded by the codon. Examples includemutations in the third position of a codon, as well in the firstposition of certain codons such as in the codon “CGG” which, whenmutated to AGG, still encodes Arg.

The terms “gene,” “recombinant gene” and “gene construct” as usedherein, refer to a DNA molecule, or portion of a DNA molecule, thatencodes a protein or a portion thereof. The DNA molecule can contain anopen reading frame encoding the protein (as exon sequences) and canfurther include intron sequences. The term “intron” as used herein,refers to a DNA sequence present in a given gene which is not translatedinto protein and is found in some, but not all cases, between exons. Itcan be desirable for the gene to be operably linked to, (or it cancomprise), one or more promoters, enhancers, repressors and/or otherregulatory sequences to modulate the activity or expression of the gene,as is well known in the art.

As used herein, a “complementary DNA” or “cDNA” includes recombinantpolynucleotides synthesized by reverse transcription of mRNA and fromwhich intervening sequences (introns) have been removed.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two nucleic acid molecules. Homology and identity can each bedetermined by comparing a position in each sequence which can be alignedfor purposes of comparison. When an equivalent position in the comparedsequences is occupied by the same base, then the molecules are identicalat that position; when the equivalent site occupied by the same or asimilar nucleic acid residue (e.g., similar in steric and/or electronicnature), then the molecules can be referred to as homologous (similar)at that position. Expression as a percentage of homology/similarity oridentity refers to a function of the number of identical or similarnucleic acids at positions shared by the compared sequences. A sequencewhich is “unrelated” or “non-homologous” shares less than 50% identity,less than 40% identity, less than 35% identity, less than 30% identity,less than 25% identity, less than 20% identity, less than 15% identityor less than 10% identity with a sequence described herein. In comparingtwo sequences, the absence of residues (amino acids or nucleic acids) orpresence of extra residues also decreases the identity andhomology/similarity.

The term “homology” may describe a mathematically based comparison ofsequence similarities which is used to identify genes with similarfunctions or motifs. The nucleic acid sequences described herein can beused as a “query sequence” to perform a search against public databases,for example, to identify other family members, related sequences orhomologs. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to nucleic acid molecules disclosed herein. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and BLAST)can be used (See www.ncbi.nlm.nih.gov).

As used herein, “identity” means the percentage of identical nucleotideresidues at corresponding positions in two or more sequences when thesequences are aligned to maximize sequence matching, i.e., taking intoaccount gaps and insertions. Identity can be readily calculated by knownmethods, including but not limited to those described in (ComputationalMolecular Biology, Lesk. A. M., ed. Oxford University Press, New York,1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data. Part1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer. Gribskov, M, and Devereux,J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman,D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determineidentity are designed to give the largest match between the sequencestested. Moreover, methods to determine identity are codified in publiclyavailable computer programs. Computer program methods to determineidentity between two sequences include, but are not limited to, the GCGprogram package (Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec.Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25:3389-3402 (1997)). The BLAST X program is publicly available from NCBIand other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIHBethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410(1990). The well-known Smith Waterman algorithm can also be used todetermine identity.

A “heterologous” region of a DNA sequence is an identifiable segment ofDNA within a larger DNA sequence that is not found in association withthe larger sequence in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene can usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is asequence where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns or syntheticsequences having codons or motifs different than the unmodified gene).Allelic variations or naturally-occurring mutational events do not giverise to a heterologous region of DNA as defined herein.

[The term “transition mutations” refers to base changes in a DNAsequence in which a pyrimidine (cytidine (C) or thymidine (T) isreplaced by another pyrimidine, or a purine (adenosine (A) or guanosine(G) is replaced by another purine.

The term “transversion mutations” refers to base changes in a DNAsequence in which a pyrimidine (cytidine (C) or thymidine (T) isreplaced by a purine (adenosine (A) or guanosine (G), or a purine isreplaced by a pyrimidine.

Nucleobases and Modified Nucleobases

The nucleobases used herein are natural nucleobases or modifiednucleobases derived 30 from natural nucleobases. Examples include, butare not limited to, uracil, thymine, adenine, cytosine, and guaninehaving their respective amino groups protected by acyl protectinggroups, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil,2,6-diaminopurine, azacytosine, pyrimidine analogs such aspseudoisocytosine and pseudouracil and other modified nucleobases suchas 8-substituted purines, xanthine, or hypoxanthine (the latter twobeing the natural degradation products). The modified nucleobasesdisclosed in Chiu et al. RNA 9: 1034-1048 (2003). Limbach et al.,Nucleic Acids Research 22: 2183-2196 (1994) are also contemplated asnucleobase moieties.

Compounds represented by the following general formulae are alsocontemplated as modified nucleobases:

In the formulae above, R⁸ is a linear or branched alkyl, aryl, aralkyl,or aryloxylalkyl group having 1 to 15 carbon atoms, including, by way ofexample only, a methyl, isopropyl, phenyl, benzyl, or phenoxymethylgroup; and each of R⁹ and RIO represents a linear or branched alkylgroup having 1 to 4 carbon atoms.

Modified nucleobases also include expanded-size nucleobases in which oneor more benzene rings has been added. Nucleic base replacementsdescribed in the Glen Research catalog (www.glenresearch.com); Krueger AT et al., Acc. Chem. Res. 40: 141-150 (2007); Kool, E T. Acc. Chem. Res.35: 936-943 (2002); Benner S. A., et al., Nat. Rev. Genet. 6: 553-543(2005): Romesberg, F. E., et al., Curr. Opin. Chem. Biol. 7, 723-733(2003, are contemplated as useful for the synthesis of the nucleic acidsdescribed herein. Some examples of these expanded-size nucleobases areshown below:

Herein, modified nucleobases also encompass structures that are notconsidered nucleobases but are other moieties such as, but not limitedto, corrin- or porphyrin-derived rings. Porphyrin-derived basereplacements have been described in Morales-Rojas, H and Kool, E T, Org.Lett. 4: 4377-4380 (2002). Shown below is an example of aporphyrin-derived ring which can be used as a base replacement:

Other modified nucleobases also include base replacements such as thoseshown below:

Modified nucleobases which are fluorescent are also contemplated.Non-limiting examples of these base replacements include phenanthrene,pyrene, stillbene, isoxanthine, isozanthopterin, terphenyl,terthiophene, benzoterthiophene, coumarin, lumazine, tethered stillbene,benzo-uracil, and naphtho-uracil, as shown below:

The modified nucleobases can be unsubstituted or contain furthersubstitutions such as heteroatoms, alkyl groups, or linking moietiesconnected to fluorescent moieties, biotin or avidin moieties, or otherprotein or peptides. Modified nucleobases also include certain‘universal bases’ that are not nucleobases in the most classical sense,but function similarly to nucleobases. One representative example ofsuch a universal base is 3-nitropyrrole.

In some embodiments, the nucleobases or modified nucleobases comprisesbiomolecule binding moieties such as antibodies, antibody fragments,biotin, avidin, streptavidin, receptor ligands, or chelating moieties.In other embodiments, the nucleobases maybe 5-bromouracil, 5-iodouracil,or 2,6-diaminopurine. In yet other embodiments, the nucleobase ismodified by substitution with a fluorescent or biomolecule bindingmoiety. In some embodiments, the substituent on the nucleobase is afluorescent moiety. In other embodiments, the substituent is biotin oravidin.

Bioprobes Comprising Nucleobases

Generally speaking, it is possible to identify sequence-specific nucleicacid segments, and to design sequences complementary to those segments,thereby creating a specific probe for a target cell, such as differentpathogen cells or mammalian cells that have, for example, mutated fromtheir normal counterparts. In principle, one can design complementarysequences to any identified nucleic acid segment. In many instances,unique sequences specific to an organism may be used as probes for aparticular organism or cell type. The quantitative phenotypic analysisof yeast deletion mutants, for example, has utilized unique nucleic acidsequence identifiers to analyze deletion strains by hybridization withtagged probes using a high-density parallel array.

Hybridization involves joining a single strand of nucleic acid with acomplementary probe sequence. Hybridization of a nucleobase comprisingbioprobe to nucleic acid sequences such as gene sequences from bacteria,or viral DNA (or RNA) offers a very high degree of accuracy foridentifying nucleic acid sequences complementary to the probe. Nucleicacid strands tend to be paired to their complements in double-strandedstructures. Thus, a single-stranded nucleobase comprising molecule willseek out its complement in a complex mixture of DNA and/or RNAcontaining large numbers of other nucleic acid molecules. Nucleobasebased bioprobe detection methods can be very specific to nucleic acidsequences. Factors affecting the hybridization or reassociation of twocomplementary nucleic acid strands include temperature, contact time,salt concentration, degree of mismatch between the base pairs, and thelength and concentration of the target and probe sequences.

The DNA, RNA, PNA, morpholino nucleic acid, or methyl phosphonatenucleic acid comprising biosensors disclosed herein may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNA or RNA fragments. In one embodiment, provided aremethods for detecting particular nucleic acid sequences in a sample. Themethod generally involves obtaining a sample suspected of containing thepolynucleotide of interest; contacting the sample with a bioprobe thatcomprises an isolated nucleobase sequence substantially complementary tothe nucleic acid sequence of interest; at least one charged amino acidattached to said nucleobase sequence; and a suitable detector, underconditions effective to allow hybridization; and detecting thehybridization. In some embodiments, the methods are used for detectingchanges in a nucleotide sequence, for example, single-nucleotidepolymorphisms or mutations. The methods generally comprise obtaining asample suspected of containing the polynucleotide of interest;contacting the sample with a bioprobe that comprises an isolatednucleobase sequence; at least one charged amino acid attached to saidnucleobase sequence; and a suitable detector, under conditions effectiveto allow hybridization; quantifying or measuring said hybridization andcomparing against a standard or control hybridization sample.

Bioprobes Comprising Peptide Nucleic Acid

DNA and RNA have a deoxyribose and ribose sugar backbone, respectively,whereas PNA's backbone is composed of repeating N-(2-aminoethyl)-glycineunits linked by peptide bonds. PNA is not known to occur naturally. Thevarious purine and pyrimidine bases are linked to the backbone bymethylene carbonyl bonds. PNAs are depicted like peptides, with theN-terminus at the first (left) position and the C-terminus at the right.

Since the backbone of PNA contains no charged phosphate groups, thebinding between PNA/DNA strands is stronger than between DNA/DNA strandsdue to the lack of electrostatic repulsion. Early experiments withhomopyrimidine strands (strands consisting of only one repeatedpyrimidine base) have shown that the Tm (“melting” temperature) of a6-base thymine PNA/adenine DNA double helix was 31° C. in comparison toan equivalent 6-base DNA/DNA duplex that denatures at a temperature lessthan 10° C. Mixed base PNA molecules are true mimics of DNA molecules interms of base-pair recognition. PNA/PNA binding is stronger than PNA/DNAbinding.

Synthetic peptide nucleic acid oligomers have been used in recent yearsin molecular biology procedures, diagnostic assays and antisensetherapies. Due to their higher binding strength it is not necessary todesign long PNA oligomers for use in these roles, which usually requireoligonucleotide probes of 20-25 bases. The main concern of the length ofthe PNA-oligomers is to guarantee the specificity. PNA oligomers alsoshow greater specificity in binding to complementary DNAs, with aPNA/DNA base mismatch being more destabilizing than a similar mismatchin a DNA/DNA duplex. This binding strength and specificity also appliesto PNA/RNA duplexes. PNAs are not easily recognized by either nucleasesor proteases, making them resistant to enzyme degradation. PNAs are alsostable over a wide pH range. Though an unmodified PNA cannot readilycross cell membranes to enter the cytosol, covalently coupling a 5 cellpenetrating peptide to a PNA can improve cytosolic delivery.

The length of the PNA probes is optimized for the specific intended use.The optimal length is a function of the distribution of purine andpyrimidine bases and in contrast to nucleotide probes is less dependenton salt concentration and pH as regulators of the stringency of thehybridization conditions.

The PNA based bioprobes may comprise one or more labelling groupsconnected to the glycine nitrogen for internal labelling of the PNAprobes or one or more labelling groups connected to one or both ends ofthe bioprobe provided that the labelling group does not destroy theperformance of the probe. As used herein, the term “label” or “labellinggroup” means a substituent, which is useful for detecting a bioprobe.

In many instances, the label is attached to the C-terminal and/orN-terminal end of the PNA bioprobe using suitable linkers. Generally,all chemical methods for N- or C-terminal labelling of peptides and for5′ or 3′ end labelling of DNA and/or RNA which are presently known mayin general terms be applied to PNAs also. For maximum stability,particularly at alkaline pH, the N-terminus of the PNA may be blocked,e.g., with an amino acid such as lysine or glycine.

Alternatively, the N-terminus may be modified by a label, an acetylgroup or by a saturated or unsaturated aliphatic group, an aromaticgroup, a heteroaromatic group, a saturated or unsaturated cyclic groupand/or a saturated or unsaturated heterocyclic group which mayoptionally be substituted by one or more heteroatom-containing groups,such as OH, NH2, SO2, SH and COOH. This type of modification preventsgradual intra-molecular rearrangement of the N-terminal residue. The useof PNA as a probe molecule has been shown previously to increase thesensitivity of biosensing assays and is particularly advantageous inelectrochemical assays because it produces lowered background currents.

PNA probes, which carry no molecular charge at neutral pH, offer manyadvantages when used for biosensing. One key advantage is the increasein binding affinity for target DNA or RNA sequences (O. Brandt, J. D.Hoheisel, Trends Biotech. 22: 617 (2004)). Moreover, when sensingschemes are used that rely on changes in surface charge upon the bindingof a nucleic acid to a sensor, lowered background signals, andconsequently improved limits of detection, can be obtained because ofthe absence of charge in the probe. Owing to this effect, ourelectro-catalytic reporter system, which relies on the attraction ofruthenium ions to the sensor surface by nucleic acids captured byimmobilized probe molecules, has been shown to exhibit bettersensitivity when PNA probes are used (Z. Fang, S. O. Kelley, Anal. Chem.81:612 (2009)).

However, there are drawbacks to the use of PNA probes. In the presentstudy, it was seen that where there are stringent requirements on thespecific sequence employed—i.e, the molecular probe must target aspecific fusion sequence—the PNA bioprobes were found not to participatein hybridization when immobilized on biosensors; moreover, precipitationout of solution of the original probe was observed. From theseobservations, it was apparent that the bioprobe sequence was prone toaggregation. We concluded that the specific sequence mandated 10 by thisparticular biosensing application was not usable when synthesized as aneutral molecule.

Ribozymes are enzymatic RNA molecules that cleave particular mRNAspecies. In certain embodiments, ribozymes capable of cleaving RNAsegments, and the resulting detection of such mRNAs or ribozymes, may beused herein with the methods and the DNA. RNA and/or PNA based bioprobesdisclosed herein.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bonds(and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids act by first binding toa target RNA. Such binding occurs through the target binding portion ofenzymatic nucleic acid which is held in close proximity to an enzymaticportion of the molecule that acts to cleave the target RNA. Thus, theenzymatic nucleic acid first recognizes and then binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cut the target RNA. Strategic cleavage of such atarget RNA will destroy its ability to direct synthesis of an encodedprotein. After an enzymatic nucleic acid has bound and cleaved its RNAtarget, it is released from that RNA to search for another target andcan repeatedly bind and cleave new targets.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis-δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. (1992);examples of hairpin motifs are described by Hampel et al. (Eur. Pat. EP0360257), Hampel and Tritz (1989), Hampel et al. (1990) and Cech et al.(U.S. Pat. No. 5,631,359); an example of the hepatitis-6 virus motif isdescribed by Perrotta and Been (1992); an example of the RNaseP motif isdescribed by Guerrier-Takada et al. (1983); and an example of the GroupI intron is described by Cech et al. (U.S. Pat. No. 4,987,071).Contemplated herein are nucleobase comprising bioprobes having aspecific substrate binding site which is complementary to one or more ofthe target gene RNA regions, and having a nucleotide sequences within orsurrounding that substrate binding site which impart an RNA cleavingactivity to the molecule.

Bioprobes Comprising Charged Functional Groups

The bioprobes disclosed herein comprise at least one charged functionalgroup attached to a nucleobase sequence capable of hybridizing to atarget molecule. In some embodiments, the charged functional group is acationic functional group, an anionic functional group or a chargedamino acid. In some embodiments, the anionic functional group is acarboxylate, a sulfate or a sulfonate. In certain embodiments, thecationic functional group is an amine or guanadinum group. In someembodiments, the charged functional group is a charged amino acid.

A charged amino acid is an amino acid that is either positively charged(protonated), or negatively charged (de-protonated) at physiological pH.Naturally occurring negatively charged amino acids include Aspartic acid(Asp, D) and Glutamic acid (Glu, E). Naturally occurring positivelycharged amino acids include Lysine (Lys, K) and Arginine (Arg, R). Anamino acid that is sometimes protonated at physiological pH is Histidine(His, H).

In addition to the above, other charged amino acids including, but notrestricted to Omithine (Om, O), Diamino-butyric acid (Dab),Diamino-propionic acid (Dap), and Aminoadipic acid (Aad). Additionalcharged amino acids are provided in table 2.

TABLE 2 Anionic Amino Acids Peptide monomer Amino Acid (Example) Fullname Structure L-aspartic acid Fmoc—Asp(OBut)—OH Fmoc-L-aspartic acidβ-t- butyl ester

D-aspartic acid Fmoc-D-Asp(OBut)—OH N-α-Fmoc-D-aspartic acid β-t.-butylester

L-glutamic acid Fmoc—Glu(OBut)—OH Fmoc-L-glutamic acid γ-t- butyl ester

D-glutamic acid Fmoc-D-Glu(OBut)—OH N-α-Fmoc-D-glutamic acid γ-t.-butylester

5-Amino Salicylic Acid Boc-5-Amino Salicylic Acid

D-γ- carboxy- glutamic acid Fmoc-D-Gla(OBut)₂—OH

L-γ- carboxy- glutamic acid Fmoc—Gla(OBut)₂—OH Fmoc-L-γ-carboxyglutamicacid γ,γ-di-t-butyl ester

4- phosphono- methyl-D- phenyl- alanine Fmoc-D-Pmp-OHFmoc-4-phosphonomethyl- D-phenylalanine

4- phosphono- methyl-L- phenyl- alanine Fmoc-Pmp-OHFmoc-4-phosphonomethyl- L-phenylalanine

L- phosphoserine Fmoc—Ser(PO(OBzl)OH)—OH N-α-Fmoc—O-benzyl-L-phosphoserine

D- phosphoserine Fmoc-D-Ser(PO(OBzl)OH)—OH N-α-Fmoc—O-benzyl-D-phosphoserine

L- phosphothreonine Fmoc—Thr(PO(OBzl)OH)—OH N-α-Fmoc—O-benzyl-L-phosphothreonine

D- phosphothreonine Fmoc-D-Thr(PO(OBzl)OH)—OH N-α-Fmoc—O-benzyl-D-phosphothreonine

O-phospho- L-tyrosine Fmoc—Tyr(PO₃H₂)—OH N-α-Fmoc—O-phospho-L- tyrosine

O-benzyl-L- phospho- tyrosine Fmoc—Tyr(PO(OBzl)OH)—OHN-α-Fmoc—O-benzyl-L- phosphotyrosine

O-benzyl- D- phospho- tyrosine Fmoc-D-Tyr(PO(OBzl)OH)—OHN-α-Fmoc—O-benzyl-D- phosphotyrosine

O-sulfo-L- tyrosine Fmoc—Tyr(SO3•NnBu₄)—OH N-α-Fmoc—O-sulfo-L- tyrosinetetrabutylammonium salt

Herein, we have generated bioprobe molecules that exhibit improved watersolubility and monolayer-forming properties with little or noaggregation. The strategy used is the introduction of a chargedfunctional group at the termini of the nucleobase sequence tobeneficially alter the probe's properties. Attachment of chargedfunctional groups, including cationic functional groups, anionicfunctional groups and charged amino acids, to PNA, DNA, RNA, or modifiednucleic acids is achieved using coupling chemistry similar to that usedin traditional PNA synthesis. This subtle modification—the inclusion ofsufficient charged functional groups—improves the behaviour of thenucleobase comprising bioprobe in that solubility in aqueous solution isimproved, and exhibited better performance when used to detect thetarget biomolecule of interest. In some embodiments, the inclusion of atleast one charged functional group results in a decrease in aggregation,as compared to the absence of the at least one charged functional group,of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%/0, atleast 85%, at least 90%, at least 95% or at least 100%. In someembodiments, the inclusion of at least one charged functional groupresults in a decrease in aggregation, as compared to the absence of theat least one charged functional group, of at least between about 10% andabout 90%, at least between about 20% and about 80%, at least betweenabout 30% and about 70%, at least between about 30% and about 90%, atleast between about 40% and about 70%, at least between about 50% andabout 80%, at least between about 50% and about 70% or at least betweenabout 60% and about 90%. In some embodiments, the inclusion of at leastone charged functional group results in a decrease in aggregation, ascompared to the absence of the at least one charged functional group, ofat least between 10% and 90%, at least between 20% and 80%, at leastbetween 30% and 70%, at least between 30% and 90%, at least between 40%and 70%, at least between 50% and 80%, at least between 50% and 70% orat least between 60% and 90° %.

Depending on the severity of the aggregation, a number of chargedfunctional groups can be used in the bioprobe. In some cases, theplacement of one or more charged functional groups, including cationicfunctional groups, anionic functional groups and charged amino acids, atthe termini of the nucleobase sequence is sufficient to achieve arobust, selective bioprobe. In certain embodiments, the number ofcharged functional groups can be as many as the total number ofnucleobases in the bioprobe sequence. The charged functional groups,including cationic functional groups, anionic functional groups andamino acids, can be positioned at one or both terminii of the nucleobasesequence, or the charged functional groups can be placed intermittentlythrough the sequence either individually, or in groups of two, three ormore, as seen in FIG. 2.

Immobilization of bioprobes on a multiarray (e.g., those of up to about100 or about 200, or about 400, or even about 1000 or so channels)sampling platform can be performed onto a transducer detection surfaceto ensure optimal contact and maximum detection. When immobilized onto asubstrate, the bioprobes are stabilized and, therefore, can be reusedrepetitively. In one illustrative embodiment, the hybridization isperformed on an immobilized target or a probe molecule attached on asolid surface such as a nitrocellulose, a nylon membrane, a glass plate,or polyvinyldifluoride (“PVDF”), a multiwell plate or another convenientsubstrate, such as the tip of a light guide, optical fiber, conductingmaterial or biosensor device, that lends itself to this purpose. In someembodiments, also included is an array device comprising multiplebioprobes affixed to a solid matrix as described herein, or suspended insolution in a linear or multidimensional format. In another aspect thebioprobes are affixed to the solid matrix in a specific array format orare placed within specific wells of a multiwell plate. Alternatively, insome embodiments, the bioprobes can be immobilized on resins,nanoparticles, nanocrystals, or microparticles.

Electrodes

In some embodiments, a nucleic acid hybridization detection assay iscarried out at a solid electrode surface. A solid electrode, such as anindium tin oxide electrode, is modified by the bioprobes describedherein, that are immobilized to the surface of the electrode. Thebioprobe hybridize with complementary target nucleic acid sequences, andthis event is detected by the reporter system

As used herein, the term “electrode” means a composition which is ableto carry or sense an electrical current or charge, and then convert itto a measurable signal. In some embodiments, an electrode is a solidsubstrate comprising a conducting material or a semiconducting material.

Electrode material can be selected according to desired redox potentialrange, ease of surface attachment of nucleic acid to surface, andappropriate or desired optical properties. As provided above, onelimitation in the selection of an electrode material is that it cannotbe identical to the material that the detection probe nanoparticlecomprises. Electrode materials include, but are not limited to, certainmetals and their oxides, such as gold, platinum, palladium, aluminum,indium tin oxide (ITO), tin oxide, fluorine-doped tin oxide, cadmiumoxide, iridium oxide, ruthenium oxide, zinc tin oxide, antimony tinoxide, platinum oxide, titanium oxide, palladium oxide, aluminum oxide,molybdenum oxide, tungsten oxide, and others. In one particularembodiment, the electrode comprises indium tin oxide (ITO).

The electrode can comprise a single conductive material or multipleconductive materials. The conductive electrode material can be layeredover a second material, such as a polymer or otherwise non-conductingsurface. In some embodiments, the electrode is formed on a solid,non-conducting substrate. The substrate can comprise a wide variety ofmaterials, including but not limited to glass, fiberglass, teflon,ceramics, silicon, mica, plastic (including acrylics, polystyrene andcopolymers of styrene and other materials), polypropylene, polyethylene,polybutylene, polycarbonate, polyurethanes, TEFLON™, combinationsthereof, and the like. Alternatively, a support can be constructed froma polymer material such as high density polyethylene (HDPE), often usedin 96-well titer plates. In yet another example, a polyacrylamide gelcan be employed as a solid support for the electrode (Dubiley et al.,(1997) Nucleic Acids Res. 25: 2259-2265).

Solid substrates on which electrodes may be formed also include printedcircuit board materials. Circuit board materials are those that comprisean insulating substrate that is coated with a conducting layer andprocessed using lithography techniques, particularly photolithographytechniques, to form the patterns of electrodes and interconnects(sometimes referred to in the art as interconnections or leads). Theinsulating substrate is generally, but not always, a polymer. As isknown in the art, one or a plurality of layers may be used, to makeeither “two dimensional” (e.g., all electrodes and interconnections in aplane) or “three dimensional” (wherein the electrodes are on one surfaceand the interconnects may go through the board to the other side)boards. Three dimensional systems frequently rely on the use of drillingor etching, followed by electroplating with a metal such as copper, suchthat the “through board” interconnections are made. Circuit boardmaterials are often provided with a foil already attached to thesubstrate, such as a copper foil, with additional copper added as needed(for example for interconnections), for example by electroplating. Thecopper surface may then need to be roughened, for example throughetching, to allow attachment of the adhesion layer.

In some embodiments, and as discussed in more detail herein, theelectrode can optionally and further comprise a passivation agent. Asused herein, the term “passivation” generally means the alteration of areactive surface to a less reactive state. Passivation can refer to, forexample, decreasing the chemical reactivity of a surface or todecreasing the affinity of a surface for nucleic acids. Stateddifferently, passivation is a method by which a surface is coated with amoiety having the ability to block subsequent binding to the surface atpoints where the moiety is bound.

In some embodiments, a passivation agent is in the form of a monolayeron the electrode surface. The efficiency of hybridization may increasewhen the detection probe is at a distance from the electrode. Apassivation agent layer facilitates the maintenance of the probe awayfrom the electrode surface. In addition, a passivation agent can serveto keep charge carriers away from the surface of the electrode. Thus,this layer can help to prevent direct physical or electrical contactbetween the electrodes and the nanoparticles of the detection probes, orbetween the electrode and charged species within the redox compoundsolution. Such contact can result in a direct “short circuit” or anindirect short circuit via charged species which may be present in thesample. Accordingly, the monolayer of passivation agents is preferablytightly packed in a uniform layer on the electrode surface, such that aminimum of “holes” exist.

In some embodiments, the electrode comprises a plurality of bioprobesattached to the electrode surface in an array format. As used herein,the terms “nucleic acid microarray,” and “nucleic acid hybridizationarray” are used interchangeably, and mean an arrangement of a pluralityof nucleobase sequences (e.g., bioprobes) bound to a support (e.g., anelectrode). The terms “addressable array” and “array” are usedinterchangeably, and mean a plurality of entities arranged on a supportin a manner such that each entity occupies a unique and identifiableposition. In the methods described herein, the entities are bioprobes(e.g., capture oligonucleotides) immobilized to the surface of anelectrode. As used herein, the terms “immobilize” and “attach” are usedinterchangeably to mean a chemical and/or mechanical association of onemoiety with one or more surfaces (e.g., solid surfaces). The associationcan be covalent or non-covalent, and can be direct or indirect.

In some embodiments, bioprobes attached to the surface of an electrodeare ordered such that each bioprobe sample has a unique, identifiablelocation on the support. The physical location on the electrode where abioprobe is attached or immobilized is referred to herein as an“attachment point.” The identity of a bioprobe bound to an electrode ata given location can be determined in several ways. One exemplary way tocorrelate a bioprobe with its location is to attach the bioprobe to thesupport at a known position (see, e.g., Pirrung, (1997) Chem. Rev. 97:473-486). Discrete locations on the support can be identified using agrid coordinate-like system. In this approach, the working area of thesupport surface can be divided into discrete areas that may be referredto interchangeably as “spots” or “patches”. Different bioprobes cansubsequently be attached to the surface in an orderly fashion, forexample, one bioprobe, or one sample of identical bioprobes, to a spot.In this strategy, the probe oligomers can be applied one or several at atime. In one exemplary method, sites at which it might be desirable totemporarily block probe binding can be blocked with a blocking agent.The blocking agent can be subsequently removed and the site freed forprobe binding. This process can be repeated any number of times, thusfacilitating the attachment of a known probe at a known location on asupport.

Localizing bioprobes to an electrode surface at known locations caninvolve the use of microspotting. In this approach, the location of thebioprobes on an electrode surface is determined by the orderedapplication of probe samples in a group. That is, bioprobes are orderedin known locations prior to application to the electrode surface. Inthis way, the location of each probe is known as it is applied.Appropriate devices for carrying out this approach are commerciallyavailable and can be used with the detection methods described herein.

As set forth above, in some embodiments a singlestranded nucleic acidsequence is used as a bioprobe. For example, a bioprobe can comprise asingle-stranded cDNA sequence complementary to a target gene of interestor to a target domain thereof. The bioprobe can be attached to theelectrode surface indirectly via an “attachment linker,” as definedherein. In this embodiment, one end of an attachment linker is attachedto a bioprobe, while the other end (although, as will be appreciated bythose in the art, it need not be the exact terminus for either) isattached to the electrode.

The method of attachment of the bioprobe to the attachment linker cangenerally be done as known in the art, and will depend on thecomposition of the attachment linker and the bioprobe. In general, thebioprobe is attached to the attachment linker through the use offunctional groups on each moiety that can then be used for attachment.Exemplary functional groups for attachment are amino groups, carboxygroups, oxo groups and thiol groups. Using these functional groups, thebioprobes can be attached using functional groups on the electrodesurface.

In one example of an attachment approach suitable for attachment ofbioprobes to an electrode surface, one or more probe capture sequencesare initially incubated with a solution of a thio-alcohol for apre-selected period of time. In some embodiments, C6 mercaptohexanol isemployed as a thio-alcohol. Thioalcohol and bioprobe are added inamounts so as to bring the final concentration of bioprobe in thesolution to about 20% or less. The incubation time permits the covalentassociation of the 3′ end of the bioprobe oligonucleotide with thehydroxyl group of the thio-alcohol. The solution is then exposed to thesurface of a support under conditions that permit association of thesulfur atom of the thio group with the surface of the support. Suitableequipment is commercially available and can be used to assist in thebinding of a target sequence to a support surface.

In another specific example, a monolayer of 12-phosphonododecanoic acidis formed on the electrode surface. The carboxylic acid of12-phosphonododecanoic acid is then activated by1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) toform an O-acylisourea intermediate. See, e.g., S. H. Brewer et al.,Langmuir (2002) 18, 6857-6865; B. L. Frey and R. M. Corn, AnalyticalChemistry (1996) 68, 3187-3193; M. Burgener et al., BioconjugateChemistry (2000) 11, 749-754; K Kerman et al., Analytica Chimica Acta(2002) 462, 39-47; E. Huanq et al., Langmuir (2000) 16, 3272-3280; andG. T. Hermanson, Bioconjugate Techniques (1996) (Academic Press: SanDiego). This activated carboxylic acid group is attacked by the primaryamine (acting as a nucleophile) of a 5′-modified C3H2 singlestranded DNAstrand to form an amide bond between the monolayer of12-phosphonododecanoic acid and the 5′ modified C3H2 ssDNA.

Other functional groups useful for attaching oligonucleotides to solidsurfaces (i.e., electrodes and nanoparticles) include, for example,moieties comprising thiols, carboxylates, hydroxyls, amines, hydrazines,esters, amides, halides, vinyl groups, vinyl carboxylates, phosphates,silicon-containing organic compounds, and their derivatives. Otherfunctional groups useful for attachment include phosphorothioate groups(see, e.g., U.S. Pat. No. 5,472,881 for the binding ofoligonucleotide-phosphorothioates to gold surfaces), aminosilanes (see,e.g., K. C. Grabar et al., J. Am. Chem. Soc. (1996) 118,1148), andsubstituted alkylsiloxanes (see, e.g. Burwell, Chemical Technology 4,370-377 (1974) and Matteucci and Caruthers, J. Am. Chem. Soc, 103,31853191 (1981) for binding of oligonucleotides to silica and glasssurfaces, and Grabar et al., Anal. Chem., 67, 735-743, for binding ofaminoalkylsiloxanes and for similar binding of mercaptoaklylsiloxanes).Oligonucleotides terminated with a 5′ thionucleoside or a 3′thionucleoside can also be used for attaching oligonucleotides to solidelectrode surfaces. The length of these attaching functional groups ischosen such that the conductivity of these molecules does not hinderelectron transfer from the nanoparticle to the electrode via thehybridized probe and target nucleic acids. Stated differently, thesefunctional groups are preferred to have higher conductivities thandouble-stranded nucleic acid.

In some embodiments, a “tag” or “linker” nucleic acid sequence can beemployed to attach bioprobes to electrode surfaces. When a tag sequenceis employed, an electrode can comprise a tag nucleic acid complement. Atag complement is a sequence that is complementary to a tag sequenceassociated with a bioprobe. Thus, when a bioprobe comprising a tagsequence is contacted with an electrode comprising a tag complementunder suitable hybridization conditions, a duplex can form.

A tag sequence can comprise, for example, a sequence that iscomplementary to a support-bound tag complement. A tag sequence can beassociated with a target sequence, which can then be amplified by PCRprior to association with a nanoparticle. The PCR amplicon will comprisea nucleic acid sequence comprising the tag sequence and a target targetsequence. The PCR amplicon then comprises a sequence that iscomplementary to a support-bound tag complement. Inclusion of a tagsequence, for example as a component of a target sequence, offers theadvantage that a support need not be specific for a given targetsequence, but rather can be universal in the sense that it is specificfor a tag complement, but not for any particular target sequence. Thus,by employing a tag complement, an electrode (or nanoparticle, asdescribed herein) can be independent of the source of a bioprobeoligonucleotide (as to species, etc.) in the sense that the electrodecan be specific for a tag sequence, but not for any particular bioprobesequence. Thus, by employing a method comprising the use of a tag-tagcomplement approach, the need to form different electrode supports fordifferent probe and/or target sequences is mitigated. See, e.g., WO94/21820, WO 97/31256, WO 96/41011 and U.S. Pat. No. 5,503,980.

Following attachment of a bioprobe to the surface of the electrode, theareas of the electrode surface to which no probe is bound can bepassivated, as defined above. A passivation process can be implementedafter probes are bound to the support, and can include sequentialsynthesis and co-deposition approaches, as is known in the art. In someembodiments, passivation is accomplished by exposing the surface tothio-alcohol, as described above. For example, the same thio-alcohol canbe used to passivate the surface as was used in attaching the probe tothe surface. In some embodiments, thio-alcohols of shorter or longerlength than those used to attach bioprobes can be employed.

In some embodiments, other molecules, i.e., “passivation moieties” canbe used passivate the surface of a support. For example, polyethyleneglycol (PEG), various alcohols and carboxylates can all be used topassivate the surface of a support, as can COO— and CONH2 moieties. Insome embodiments, passivation moieties can also be non-covalently orcovalently attached. Indeed, virtually any material can be used topassivate a support surface, with the understanding that the passivationmaterial must associate with the support to form a protective layercoating the support, and that the passivating process, which can beperformed after a probe is already associated with the surface of thesupport, does not damage any probes already bound to the support. Asdescribed above, a passivation step can also be performed to reduce thepotential for nonspecific association between a nanoparticle complex anda support.

In analyzing a liquid sample using electrodes and electronic equipmentand techniques, the size and spacing of electrodes can affect whetherdiffusion of an analyte through the sample to an electrode occurs by aplanar or non-planar path. Micro-electrode arrays are of a size andspacing such that in detecting chemical species of a solution, thespecies will diffuse toward or approach an electrode of themicro-electrode array in a non-planar fashion, e.g., in a curved orhemispherical path of diffusion. In contrast, non-microclectrodes, i.e.,“macro-electrodes,” cause diffusion of an analyte through a soluteaccording to a substantially planar path. It is also understood thatsome electrode configurations can cause diffusion to take place by a mixof planar and non-planar paths, in which case the electrodes can beconsidered a micro-electrode array, especially if the diffusion occurspredominantly (e.g., greater than 50%) according to a non-planar path,or if the size of the electrodes is less than 100 μm.

The electrodes of a micro-electrode array are positioned near each otherin an arrangement that will result in non-planar diffusion as described.The arrangement of the electrodes can be any arrangement that results insuch diffusion, with a working and a counter electrode beingsubstantially evenly spaced from each other. One electrode may bearranged into a shape or figure or outline that will produce intersticeswithin which the second electrode may be placed. For instance, oneelectrode can be arranged as an increasing radius, substantiallycircular spiral, with a continuous, long and narrow interstitial areabeing created between each successively larger revolution of electrode.The other electrode can be positioned in the interstitial area betweenrevolutions, while the electrodes remain insulated from one another. Thewidth and spacing of the electrodes can be arranged to result inmicro-electrode array performance.

According to other forms of such micro-electrode arrays, the spiral maynot be substantially circular, but could include linear, square, angled,or oblong or oval features. Or, the electrodes could be arranged in anyother geometric form whereby the electrodes are placed adjacent to eachother and within the other's respective interstitial area, e.g., byfollowing a similar path separated by a substantially uniform gap.

In one particular embodiment, the micro-electrode can be arranged intoan interdigitated array, meaning that at least a portion of electrodeelements of the working electrode are placed substantially parallel toand in alternating succession with at least a portion of the electrodeelements of the counter electrode, e.g., in an alternating,“finger-like” pattern. Such interdigitated micro-electrode arraysinclude electrode elements (sometimes referred to as “fingers”) and acommon element (“contact strip”) which commonly connects the electrodeelements.

The electrodes and their components can be of dimensions, meaning thewidth of the electrode components as well as the separation betweencomponents that can provide an array with useful properties. e.g.,useful or advantageous capabilities with respect to contacting asubstance or measuring electrical properties. Advantageously,interdigitated arrays can be prepared at dimensions that allow forcontact with and measurement of electrical properties of a relativelysmall sample of a substance.

The thickness of the electrode components can be sufficient to support adesired electric current. Exemplary thicknesses can be in the range fromabout 20 to 200 nanometers (nm), with an exemplary thickness being about10-50 nm.

The electrodes can independently have a number of interdigitatedelectrode elements sufficient to provide utility, e.g., allowing contactwith a substance to measure its electrical behavior. Conventionally, thearray can have substantially the same number (equal, plus or minus one)of electrode elements in the working electrode as are in the counterelectrode, allowing the electrode elements to be paired next to eachother in an alternating sequence. In some embodiments of the array, suchas in some of the applications described below for electrochemicalsensors, each electrode of an array may typically have from about 4 toabout 30 electrode elements.

An exemplary electrode is a nanostructured microelectrode (NME). NMEsare electrodes, which are nanotextured and thus have an increasedsurface area. NMEs of the above-described materials are highlyconductive and form strong bonds with the bioprobes. Exemplary NMEs havea height in the range of about 0.5 to about 100 microns (μm), forexample in the range of about 5 to about 20 microns. (e.g. 10 microns);a diameter in the range of about 1 to about 10 microns; and havenanoscale morphology (e.g., are nanostructured on a length scale ofabout 1 to about 300 nanometers and more preferably in the range ofabout 10 to about 20 nanometers). NMEs can be any of a variety ofshapes, including hemispherical, irregular (e.g., spiky), cyclical(wire-like) or fractal (e.g., dendritic). The surface of an NME may befurther coated with a material, which maintains the electrode's highconductivity, but facilitates binding with a probe. For example,nitrogen containing NMEs (e.g., TiN, WN or TaN) can bind with an aminefunctional group of the probe. Similarly, silicon/silica chemistry aspart of the NME can bind with a silane or siloxane group on the probe.

Reporter Detection Systems

Reporter systems may include chemical or biological reporters. In someembodiments, the reporter system may be an enzyme fragment, (see, e.g.,US Patent Application No. 2007/0105160, incorporated by reference hereinin its entirety), a protein (e.g., c-myc or other tag protein orfragment thereof), an enzyme tag, a fluorescent tag, a fluorophore tag,a chromophore tag, a Raman-activated tag, a chemiluminescent tag, aquantum dot marker, an antibody, a radioactive tag, or a combinationthereof. In some aspects, enzyme activity is monitored before and aftertreatment to determine if enzyme activity is modulated by treatment withtest compounds or agents. In other aspects, fluorescent activity ismonitored before and treatment to determine if marker levels, forexample, FRET-induced fluorescent levels, are modulated by treatmentwith test compounds or agents.

In some embodiments, the fluorescent tag includes a fluorescent dye orfluorophore. A diversity of fluorophores with a distinguishable colorrange allows visualization of multiple targets in parallel. Examples offluorescent dyes developed as labels include fluorescein and rhodaminedyes (collectively called xanthene dyes). For example, fluorescein typedyes, such as FAM, JOE, HEX and NED are used for preparing real-time PCRprobes, or so-called TaqMan® probes (Holland et al., Proc. Natl. Acad.Sci. USA 88, 7276(1991); Lee et al., Nucleic Acids Res. 21, 3761(1993).Likewise, various rhodamine dyes have been used for preparing real-timePCR probes based on oligonucleotides homo-doubly labeled with twoidentical dyes (Mao, et al., US patent application No. 20050272053).Fluorescently labeled antibodies are important tools in fluorescenceimmunochemistry-based detections, and fluorescein and rhodamine dyeswere among the first dyes used for preparing antibody conjugates.However, many of these xanthenes dyes suffer from problems offluorescence quenching and poor water solubility.

Xanthene dyes which absorb and emit in a variety of colors may also beused as a reporter system. Xanthene dyes with short wavelengthabsorption/emission include, for example, fluorescein and rhodamine 110and sulfonated rhodamine 110. Xanthenes dyes with longer wavelengthabsorption/emission profile include the fluorescein derivative JOE,which has a methoxy substituent at the 4 and 5-positions, respectively,has absorption/emission maxima at 520/548 nm, compared to the parent dyefluorescein (or FAM), which has absorption/emission at 495/520 nm. Therhodamine dye ROX has absorption/emission at 575/602 nm, compared to theparent rhodamine dye carboxy-rhodamine 110, which absorbs and emits at502/524 nm. Additional dyes are described by Sauer, et al. Journal ofFluorescence 5(3), 247(1995), David, et al. Tetrahedron Letters 49(11),1860(2008) and Liu, et al. Tetrahedron Letters 44, 4355(2003).

Electrocatalytic Reporter Groups

The redox reporter can be a redox-active metal center or a redox-activeorganic molecule. It can be a natural organic cofactor such as NAD.NADP. FAD or a natural metal center such as Blue Copper, iron-sulfurclusters, or heme, or a synthetic center such as an organometalliccompound such as a ruthenium complex, organic ligand such as a quinone,or an engineered metal center introduced into the protein or engineeredorganic cofactor binding site. Cofactor-binding sites can be engineeredusing rational design or directed evolution techniques. The redoxreporter can be bound covalently or non-covalently to the protein,either by site-specific or adventitious interactions between thecofactor and protein. It can be intrinsic to the protein such as a metalcenter (natural or engineered) or natural organic (NAD, NADP, FAD) ororganometallic cofactor (heme), or extrinsic (such as a covalentlycoupled synthetic organometallic cluster). The redox reporter can be,for example, linked (e.g., covalently) to a residue on the proteinsurface.

The redox reporter can be a metal-containing group (e.g., a transitionmetal-containing group) that is capable of reversibly or semi-reversiblytransferring one or more electrons. A number of possible transitionmetal-containing reporter groups can be used. Advantageously, thereporter group has a redox potential in the potential window below thatsubject to interference by molecular oxygen and has a functional groupsuitable for covalent coupling to the protein (e.g., thiol-reactivefunctionalities such as maleimides or iodoacetamide for coupling tounique cysteine residues in the protein). The metal of the reportergroup should be substitutionally insert in either reduced or oxidizedstates (i.e., advantageously, exogenous groups do not form adventitiousbonds with the reporter group). The reporter group can be capable ofundergoing an amperometric or potentiometric change in response toligand binding. In one embodiment, the reporter group is water soluble,is capable of site-specific coupling to a protein (e.g., via athiol-reactive functional group on the reporter group that reacts with aunique cysteine in the protein), and undergoes a potentiometric responseupon ligand binding. Suitable transition metals for use herein include,but are not limited to, copper (Cu), cobalt (Co), palladium (Pd), iron(Fe), ruthenium (Ru), rhodium (Rh), osmium (Os), rhenium (Re), platinum(Pt), scandium (Sc), titanium (Ti), vanadium (V), chromium (Cr),manganese (Mn), nickel (Ni), molybdenum (Mo), technetium (Tc), tungsten(W), and iridium (Ir). That is, the first series of transition metals,the platinum metals (Ru, Rh, Pd, Os, Ir and Pt), along with Fe, Re, W,Mo and Tc, may be used. Exemplary metals that do not change the numberof coordination sites upon a change in oxidation state, includeruthenium, osmium, iron, platinum and palladium.

The reporter group can be present in the biosensor as a covalentconjugate with the protein or it can be a metal center that forms partof the protein matrix (for instance, a redox center such as iron-sulfurclusters, heme, Blue copper, the electrochemical properties of which aresensitive to its local environment). Alternatively, the reporter groupcan be present as a fusion between the protein and a metal bindingdomain (for instance, a small redox-active protein such as acytochrome). Preferably, the reporter group is covalently conjugated tothe protein via a maleimide functional group bound to a cysteine (thiol)on the protein. In any case, the reporter group is attached to theprotein so that it is located between the protein and the electrode.

In the instant embodiment, to transduce nucleic acids hybridization intoan electrical signal, an electrocatalytic reporter system previouslydeveloped by our laboratory is used (Lapierre, M. A. et al., Anal. Chem.2003, 75, 6327-6333). This reporter system relies on the accumulation of[Ru(NH₃)₆]³⁺ at electrode surfaces when polyanionic species like nucleicacids bind, and the catalysis of the reduction of Ru(III) via theinclusion of [Fe(CN)₆]³⁻, which regenerates Ru(III) and allows multiplereductions per metal center. When PNA-modified NMEs were challenged witha complementary sequence, detectable signal changes could be clearlydetected through the femtomolar concentration range. Negligible signalchanges were observed with completely non-complementary sequences.

Nucleic Acid Targets

A target sequence can be selected on the basis of the context in whichthe present methods are employed. Target sequences can vary widely. Forexample, desirable target sequences include, but are not limited, tocharacteristic or unique nucleic acid sequences found in variousmicrobes or mutated DNA that can be used in the diagnosis of diseases,in environmental bioremediation, in the determination of geneticdisorders, and in genetic epidemiology.

Functional equivalents of known sequences can also be used as targetsequences.

The target sequence may be a portion of a gene, a regulatory sequence,genomic DNA, cDNA, RNA including mRNA and rRNA, or others. The targetsequence can be a target sequence from a biological sample, as discussedherein, or can be a secondary target such as a product of anamplification reaction. The target sequence can take many forms. Forexample, a target may be contained within a larger nucleic acidsequence, i.e., all or part of a gene or mRNA, a restriction fragment ofa plasmid or genomic DNA, among others. Target nucleic acids can beexcised from a larger nucleic acid sample using restrictionendonucleases, which sever nucleic acid sequences at known points in anucleic acid sequence. Excised nucleic acid sequences can be isolatedand purified by employing standard techniques. Target sequences can alsobe prepared by reverse transcription processes. See, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y.(1992)). A target sequence can comprise one or more different targetdomains. A target domain is a contiguous, partial sequence (i.e., asub-sequence) of the entire target sequence, and may be any nucleotidelength that is shorter than the entire target sequence. In someembodiments, a first target domain of a target sequence hybridizes acapture probe, while a second and different target domain hybridizes anoligonucleotide component of a detection probe. Target domains may beadjacent or separated, as indicated. For example, a first target domaincan be directly adjacent (i.e., contiguous) to a second target domain,or the first and second target domains may be separated by anintervening target domain. Assuming a 5′ to 3′ orientation of a targetsequence, a first target domain may be located either 5′ to a secondtarget domain, or 3′ to a second domain.

If desired, a target sequence may further comprise an additional moietysuch as one partner of a ligand-binding pair, in order to facilitatebinding to a detection probe comprising a nanoparticle attached to theother partner of the ligand-binding pair. For example, the targetsequence may comprise a biotin moiety, which will facilitate binding toa detection probe comprising a nanoparticle attached to streptavidin.The biotin moiety may be incorporated into the target sequence usingamplification methods that are analogous to known methods used toincorporate fluorescent moieties into target molecules, as set forth inmore detail below. Nucleic acid sequences of any practical length can beused as a target sequence. Generally, a target sequence is between 10and 50 nucleotides in length, and thus target sequences of 10, 15, 20,25, 30, 35, 40, 45 or more nucleotides can be employed. However, targetsequences of any length can be employed in the methods disclosed herein,and in some cases, may be shorter than ten nucleotides and longer than50 nucleotides. For example, target sequences may be about 60nucleotides long, about 75 nucleotides long, about 85 nucleotides long,about 100 nucleotides long, about 200 nucleotides long, about 300nucleotides long, about 400 nucleotides long, about 500 nucleotideslong, or even longer. In some instances, the target sequence may bebetween 60-500 nucleotides long. If desired by the artisan, a targetsequence may be fragmented prior to hybridization steps by usingenzymatic, mechanical or other means as known in the art.

In some embodiments, target sequences can be isolated from biologicalsamples, including, but not limited to, bodily fluids (e.g., blood,urine, serum, lymph, saliva, anal and vaginal secretions, perspirationsemen, etc., of virtually any organism); environmental samples (e.g.,air, plant, agricultural, water and soil samples), and research samples(i.e. amplification reaction products, purified samples such as purifiedgenomic nucleic acids, and unpurified samples of bacteria, virus,genomic DNA, etc.).

If required, the target nucleic acid can be isolated from sourcebiological samples using known techniques. For example, samples can becollected and concentrated or lysed, as required. Appropriate adjustmentof pH, treatment time, lytic conditions and sample modifying reagentscan also be made in order to optimize reaction conditions. Suchmodification techniques are well known to those of skill in the art.Methods for nucleic acid isolation and purification can comprisesimultaneous isolation of, for example, total nucleic acid, or separateand/or sequential isolation of individual nucleic acid types (e.g.,genomic DNA, cDNA, organelle DNA, genomic RNA, mRNA, polyA+ RNA, rRNA,tRNA) followed by optional combination of multiple nucleic acid typesinto a single sample.

Methods for nucleic acid isolation can optionally be optimized topromote recovery of pathogen-specific nucleic acids. In some organisms,for example fungi, protozoa, grampositive bacteria, and acid-fastbacteria, cell lysis and nucleic acid release can be difficult toachieve using general procedures, and therefore a method can be chosenthat creates minimal loss of the pathogen subset of the sample.Semi-automated and automated extraction methods can also be used fornucleic acid isolation.

In some embodiments, a target nucleic acid comprises a double-strandednucleic acid. Double stranded nucleic acid sequences can be prepared,for example, by isolating a double stranded segment of DNA.Alternatively, multiple copies of single stranded complementaryoligonucleotides can be synthesized and annealed to one other underappropriate conditions. In order to provide a singlestranded target forhybridization, double-stranded nucleic acids are preferably denaturedbefore hybridization. The term “denaturing” refers to the process bywhich strands of oligonucleotide duplexes are no longer base-paired byhydrogen bonding and are separated into single-stranded molecules.Methods of denaturation are well known to those skilled in the art, andinclude thermal and alkaline denaturation. RNA isolation methods areknown to one of skill in the art. See, Albert et al. (1992) J Virol66:5627-2630; Busch et al. (1992) Transfiusion 32:420-425; Hamel et al.(1995) J Clin Microbiol 33:287-291; Herrewegh et al. (1995) J ClinMicrobiol 33:684-689; Izraeli et al. (1991) Nuc Acids Res 19:6051;McCaustland et al. (19911 Virol Methods 35:331342; Nataraian et al.(1994) PCR Methods Appl 3:346-350; Rupp et al. (1988) BioTechniques6:56-60; Tanaka et al. (1994) Gen Virol 75:2691-2698; and Vankerckhovenet al. (1994) J Clin Microbiol 30:750-753.

Targeting mRNA is attractive given that most mRNAs exist as linearsequences and therefore denaturation is not required. Multiple copies ofmRNAs are typically present within a cell, providing built-inamplification at the cellular level, improving the prospects for thedirect analysis of small numbers of cells. However, the large sizes ofmRNAs present a challenge for chipbased sensing as the slow diffusion ofsuch large molecules can impede rapid analysis.

To aid in the detection of of large molecules, we have increased thereach of the sensor into the solution volume, thus providing greaterinteraction between the target molecules in solution and the bio-probemolecules tethered to the sensor surface. Using existing diffusionalmodels, (P. E. Sheehan et al., Nano Lett. 2005, 5, 803; and

P. R. Nair, et al., Nano Lett. 2008, 8, 1281) and taking intoconsideration the copy number of the target mRNA and its rate ofdiffusion, when the spatial footprint of our sensors is increased to−100 microns, we can detect as few as 10 cellsexpressing the target mRNAwithin analysis times approaching 30 minutes (a volume of 30 microliterswas used in this calculation to yield a concentration of 1.7 fM for themRNA)

In some embodiments, an increase of about 10% to about 20% in signal, ofabout 10% to about 40% in signal, or of about 1% to about 20% in signalas compared to a control signal in the absence of target nucleotide, isobtained when the bioprobe hybridizes to a target nucleotide. In certainother embodiments, the increase is about 30%. In certain embodiments,the increase in signal is about 40% when the bioprobe is hybridized to atarget nucleotide. In some embodiments the increase is about 50% toabout 60%. In certain embodiments, the increase in signal is about 70%,about 80%, about 90%, about 100%, about 125%, about 150% A, about 175%,or about 200% when the target nucleic acid is hybridized to thebioprobes disclosed herein.

In some embodiments, a decrease of about 10% to about 20% in signal isobtained when the bioprobe hybridizes to a target nucleotide. In certainother embodiments, the decrease is about 30%. In certain embodiments,the decrease in signal is about 40% when the bioprobe is hybridized to atarget nucleotide. In some embodiments the decrease is about 50% toabout 60%. In certain embodiments, the decrease in signal is about 70%,about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, orabout 200% when the target nucleic acid is hybridized to the bioprobesdisclosed herein.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the embodiments. The specificmethods exemplified can be practiced with other species. The examplesare intended to exemplify generic processes.

EXAMPLES Example 1: General Design Strategy for Bioprobe

Herein, bioprobe molecules are disclosed that exhibit improved watersolubility and monolayer-forming properties with substantially no orlittle aggregation detected that can appreciably interfere with bindingof the disclosed bioprobe molecules with a target nucleic acid sequence.Introduction of charged functional groups at the termini of thenucleobase sequence can beneficially alter the probe's properties,including solubility profiles. One exemplary embodiment is theattachment of charged amino acids to PNA, DNA and RNA using similarcoupling chemistry to that used in traditional PNA synthesis. Forexample, the inclusion of sufficient charged amino acid units mayimprove the behaviour of the nucleobase comprising bioprobe in thatsolubility may be improved in aqueous solution, and may exhibit betterperformance when used to detect the target biomolecule of interest. Incase of persistent solubility or aggregation issues, a multitude ofcharged amino acids may be used. The amino acids may be placed at thetermini as shown in Table 3 below.

TABLE 3 Sample Bioprobe sequences Probe No Sequence SEQ ID NO 1Cys-Gly-NB-Xaa  1 & 16 2 Cys-Gly-Xaa-NB-Xaa  2 & 16 3 Cys-Gly-Xaa-Xaa-NB 3 4 Cys-Gly-Xaa-Xaa-Xaa-NB-Xaa  4 & 16 5 Cys-Gly-Xaa-Xaa-Xaa-NB-Xaa-Xaa 5 & 17 6 Cys-Gly-Xaa-Xaa-Xaa-NB-Xaa-Xaa-Xaa  6 & 18 7Cys-Gly-Xaa-NB-Xaa-NB  7 & 16 8 Cys-Gly-Xaa-NB-Xaa-NB-Xaa-NB-  8 & 16 &16 9 Cys-Gly-Xaa-NB-Xaa-Xaa-NB-Xaa-  9 & 17 & 16 10Cys-Gly-Xaa-Xaa-NB-Xaa-NB-Xaa-Xaa 10 & 16 & 17wherein Xaa is a charged amino acid such as Asp, Glu. Aad, Lys, Om, Dab,Dap, Arg or an amino acid selected from Table 2 and NB is anoligonucleobase sequence such as a DNA, PNA or RNA. The charged aminoacids may be replaced with the charged functional groups disclosedherein, including anionic functional groups such as carboxylate, sulfateor sulfonate, and cationic functional groups such as amine orguanadinum. In some embodiments, the charged functional groups areplaced intermittently through the nucleobase sequence without adverselyaffecting the ability of the nucleobase sequence to hybridize with thetarget. FIG. 2D shows different strategies for placement of chargedamino acids in conjugation with a 20 nucleobase bioprobe sequence.

Example 2: Target Gene

bcr-abl gene fusion is specific to chronic myleoid leukemia (CML) as aninteresting model system to hone the capabilities of our chip-basedsensors so that small numbers of cancer cells could be analyzed. Thebcr-abl mRNA transcript is a RNA molecule that is 8500 nucleotides long(F. vanRhee, et al., Blood 1996, 87, 5213-5217).

As described supra, using existing diffusional models, and taking intoconsideration the copy number of the bcr-abl fusion mRNA (˜3000 copiesper cell) (M. Wilda. et al., Oncogene 2002, 21, 5716) and its rate ofdiffusion, it was determined that increasing spatial footprint of oursensors to 100 microns, would result in detection of as few as 10 CMLcells within analysis times approaching 30 minutes (a volume of 30microliters was used in this calculation to yield a concentration of 1.7fM for the mRNA).

Example 3: Biosensor Design

Previous work on chip-based microsensors focused on palladium sensorsthat were 5-10 microns in diameter (L. Soleymani, et al., Nat.Nanotechnol. 2009, 4, 844). To extend the reach of these sensors intosolution, it was necessary to use gold as the electrode material.Electroplated palladium did not produce grain sizes that allowed growthof the sensor into solution, and instead the sensors grew along thesurface of the chip. In contrast, the electroplating of gold producedspiky structures with large substructures extending many microns intosolution when low plating potentials were used (FIG. 5). After extensivevariation of plating conditions (including plating potential, supportingelectrolyte, and plating time), biosensors with the desired 100 micronfootprint were obtained (FIG. 1B).

In order to test these biosensors against the bcr-abl mRNA target,bioprobes that would specifically bind to the junction region betweenthe two fused genes were required. Initial efforts to produce afunctional probe for this region (FIG. 2) included the synthesis of DNAand PNA bioprobes. When challenged with mRNA from the K562 cell line,which contains the most common form of the bcr-abl gene fusion,hybridization was not detected with either DNA bioprobe or PNA bioprobe.Hybridization analysis was carried out by monitoring reductive currentsin a solution containing a Ru(III)/Fe(III) electrocatalytic reportersystem. The solution used in these trials contained 1 ng/μl of totalmRNA, which contained >10,000,000 copies of the target per microliter,or >20 μM—a concentration that should have been readily detectable basedon our calculations. The DNA bioprobe modified biosensors exhibited highbackground signals and small decreases in current upon introduction ofthe mRNA solution. The PNA-modified biosensor exhibited a much lowerbackground current, but here too, only small decreases in current wereobserved upon hybridization with mRNA rather than the expected increase.

Example 4: Bioprobe Design

Since the PNA and DNA based bioprobes failed in functioning as desired,developed herein are a new class of probe molecules: amino acid/nucleicacid chimeras (ANAs) that were necessary in order to detect a specificcancer biomarker. ANAs overcome three fundamental limitations that wefound arise when using neutral probe molecules: poor solubility,aggregation, and poor monolayer quality. The resulting sensor systemreported herein displays excellent sensitivity and specificity.Remarkably, it achieves this excellent performance, requiring a single,simple cell lysis step prior to analysis, even when analyzing unpurifiedsamples.

The introduction of charged amino acids at the termini of the PNAsequence described supra beneficially altered the PNA probe'sproperties. Attachment of amino acids to PNAs was achieved usingidentical coupling chemistry to that used in traditional PNA synthesis.

The inclusion of 2 aspartic acid units—rectified the behaviour of ourprobe in that it was now highly soluble in aqueous solution, andexhibited much better performance when used to detect the bcr-abl mRNA.Low background currents were observed at probe-modified sensors, andsignificant increases occurred upon hybridization of K562 mRNAcontaining the fusion (FIG. 2C).

Example 5: Experimental Materials and Methods

Chemicals and materials. Gold (III) chloride (99.9%),hexaammineruthenium (III) chloride, potassium ferricyanide, magnesiumchloride, and 6-mercapto-1-hexanol (97%), 10×TBE buffer, UltraPure™Agarose, dimethylformamide, piperidine, TFA, m-cresol, TIPS, diethylether, acetonitrile, DTT were purchased from Sigma-Aldrich Canada Ltd.;70% perchloric acid, sulfuric acid, ACS-grade acetone, and isopropylalcohol were obtained from EMD; 6N hydrochloric acid was purchased fromVWR. PNA monomers were purchased from Link technologies, Knorr resin waspurchased from NovaBiochem; HATU and N-methylmorpholine were purchasedfrom Protein Technologies. Inc., and RedSafe™ from FroggaBio: K562 cellline was obtained from the ATCC, 25-mL suspension flasks were purchasedfrom Sarstedt, Iscove's Modified Dulbecco culture medium and fetalbovine serum were obtained from Invitrogen; CML patient samples wereprovided by Dr. Minden of Princess Margaret Hospital, whole blood wasobtained at the Princess Margaret Hospital blood laboratory.

Preparation and purification of oligonucleotides. ANA and PNAoligonucleotides were synthesized using the solid-phase synthesisapproach on a Prelude automated peptide synthesizer (ProteinTechnologies, Inc.). ANA probe corresponding to el3a2:NH₂-Cys-Gly-Asp-TGAAGGGCTTCTTCCTTATT-Asp-CONH2 (SEQ ID NO: 11) and ANAprobe corresponding to el4a2:NH₂-Cys-Gly-Asp-TGAAGGGCTITTGAACTCTG-Asp-CONH₂ (SEQ ID NO: 12). PNAprobes contained the same sequence but lacked the Asp residues. DNAprobe was commercially purchased and a thiol containing linker was addedin-house: TGAAGGGCTTTTGAACTCTG-linker-SH (SEQ ID NO: 13). Negativecontrol probe was: NH₂-Cys-Gly-Asp-ATCTGCTCTGTGGTGTAGTT-Asp-CONH₂ (SEQID NO: 14). All probe molecules were stringently purified using anAgilent 1100 series HPLC. Concentration was determined by measuringabsorbance at 260 nm.

Chip fabrication. The chips were produced at the Canadian PhotonicsFabrication Center. Silicon wafers were passivated using a thick layerof thermally grown 2 micron silicon dioxide. A 350-nm gold layer wasdeposited on the chip using electron-beam-assisted gold evaporation. Thegold film was patterned using standard photolithography and a lift-offprocess. Using chemical vapour deposition a 500-nm layer of insulatingsilicon dioxide was deposited. Five (5) micron circular apertures and2×2 mm bond pads were exposed on the electrodes through the top layerusing standard photolithography.

Fabrication of microelectrodes. The chips were washed in acetone, rinsedwith isopropyl alcohol and then deionized water and briefly dried with aflow of nitrogen. All electrodeposition was performed at roomtemperature with a Bioanalytical Systems Epsilon potentiostat with athree-electrode system containing an Ag/AgCl reference electrode and aplatinum wire auxiliary electrode. The 5 micron apertures on the chipswere used as the working electrode and were contacted using the exposedbond pads. Electrodeposition of gold microelectrodes were accomplishedby dipping the chip into the plating solution (20 mM H₂AuC₁₄ in 0.5Mhydrochloric acid) and applying constant of 0 mV for 175 seconds.

Modification of microelectrodes. A solution containing 5 μM thiolatedPNA probe in 50 mM sodium chloride was added to the sensors and left ina dark humidity chamber overnight at room temperature for self-assemblyof a monolayer. A solution of 10 μM MCH was then added to each chip for1 hour at room temperature to block the bare surface of the NME. Thechip was then washed twice with 50 mM NaCl.

Electrochemical measurements. Electrochemical signals were measured in asolution containing 10 μM [Ru(NH₃)₆]³⁺ and 2 mM [Fe(CN)₆]³⁻ in 1×PBS.Differential pulse voltammetry signals before and after hybridizationwere collected with a scan rate of 100 mV s−1 and scanned from 0 mV to−350 mV. Results were quantified by subtracting peak currents in DPVscans as follows, ΔI=Iafter hybridization-Ibefore hybridization.

Cell culture. K562 cells were cultured in 25 mL suspension cell flaskswith vent caps in Iscove's Modified Dulbecco's Medium/10% fetal bovineserum. The cells were grown in a humidified incubator (70-95%/o) at37.0° C. with CO₂ (5%). Cultures were maintained by the replacement byfresh medium every 2 to 3 days. Subculture was performed when the cellpopulation reached 500.000 cells/mL. K562 cells and patient samplespreparation. K562 cells were collected and centrifuged at 600 ref for 5minutes at 4° C. The media was then removed and the cells were washedwith equal volume of 1×PBS. The cell pellet was then resuspended in1×PBS and used for lysis. CML patient samples frozen stock were thawedquickly at 37° C. in a water bath. Immediately, the cells were added to10 mL of fresh media supplemented with 10% FBS and centrifuged at 400ref for 5 minutes at 4° C. The pellet was washed fresh media and thepellet was resuspended in 1×PBS for further use. Primary samples werecollected following informed consent according to an REB approvedprotocol.

mRNA isolation. Total mRNA was isolated from cells using Dynabeads®(Invitrogen). Quality of the mRNA sample was tested using 1% agarose gelelectrophoresis.

Cell lysis. Cells lysis (K562, patient samples, whole blood) wasachieved using an electrical lysis chamber. Pt wires used to produce theelectric field were inserted into PDMS (polydimethylsiloxane) membrane.The channels for the cell solution to flow through were made with dullend needle and were vented with N₂ for 1 hour prior to use. Resuspendedcells were taken into a 5 mL syringe and loaded into a syringe pump. Incase of whole blood, it was diluted 100 times in IX PBS and 1 mL wasloaded into a 5 mL syringe. Lysis was achieved at a flow rate 25 μL/min,400 V and 1 mA current.

Hybridization protocol. Hybridization solutions contained either totalmRNA, or unpurified cell lysate in 50 mM NaCl. Electrodes were incubatedwith the target sequences at 37° C. in a humidity chamber in the darkfor 30 minutes and were washed extensively twice with 50 mM NaCl priorto electrochemical analysis. Hybridization volume was typically 30 μL.

Example 6: Biosensor Use in CML Detection

With optimized biosensors and chimeric amino acid/nucleic acid (ANA)probes in hand, the limit of detection and dynamic range was determinedwhen the system was challenged with purified cellular mRNA. A controlprobe with an unrelated sequence was monitored alongside the bcr-ablprobe and showed no signal change even at the highest concentrationtested, indicating that the assay had excellent specificity. Detectablesignal was observed as low as 1 pg/L of total mRNA (FIG. 3) and thesignal increased linearly over four logs of target concentration. Thisresult is comparable with a commercially available polymerase chainreaction (PCR) assay designed to specifically detect CML; the assay hasa similar detection limit (Z. Jobbagy, et al., Mol. Diagnostics, 2007,9, 220). This chip-based system is the first ever to exhibit PCR-likesensitivity.

While PCR is a highly sensitive technique for sequence detection, itoften requires extensive sample processing and nucleic acid purificationto eliminate interferents that inhibitors of the enzymes used foramplification. Given that our detection system does not rely on anyenzymatic reactions, it is much more tolerant of unpurified samples.

To prove detection fusion mRNA from an unpurified sample, K562 cellswere lysed using an electric field. Rapid lysis was achieved (under 5minutes) without the use of added reagents. Lysates were generatedcontaining 10 to 1000 cells; the results of their incubation withbcr-abl probe-modified sensors are illustrated in FIG. 4A. The negativecontrol was a half-complementary probe (a probe for the e13a2 genefusion) and was found to show no signal increase, confirming thespecificity of the assay. The detection of 10 cells—present as anunpurified lysate—indicates that these biosensors are highly sensitiveand robust.

Leukocytes from CML patients were then analysed using the samelysis-only sample preparation procedure and the sensor and assaydescribed above. The negative control was a probe only halfcomplementary to the bcr-abl gene fusion. Absence of a positive signalwith the negative control again confirmed the specificity ofhybridization. Here the detection limit approached 100 cells, owing tothe fact that the sample contained both CML cells and normal leukocytes.

Example 7: CML Detection in Blood Samples

CML samples in whole blood were analysed. Analysis of complex samples ischallenging due to their heterogeneity: in particular, direct analysisin blood may be impeded by rapid degradation of nucleic acids bynucleases, and by the fouling of surfaces by the components of blood.Blood spiked with CML cells was used to determine whether the analysisof whole blood samples would be feasible. An offset of the backgroundsignal was observed when blood only was used. The use of a control probewas introduced to follow this shift, which remained constant whensamples with varying numbers of K562 cells were analyzed. By referencingmeasurements for the target-probe case to the control-probe case, as fewas 10 K562 cells were detected even in the presence of a 5,000.000-foldexcess of blood cells (FIG. 3E).

This study represents the first report of highly sensitive, specificbioprobe for analysis of nucleic acid biomarkers in relevant samples. Itrelies on direct lysis of the cells under study within amedium—blood—that comprises a huge excess of noncomplementary molecules.Specifically, the biosensors described herein achieved detection of mRNAderived from 10 cells present as an unpurified, unprocessed lysate.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A bio-probe comprising: a nucleobase sequence capable of hybridizingto a target nucleotide; at least one charged functional group attachedto said nucleobase sequence, wherein said charged functional groupcomprises a cationic functional group, an anionic functional group, acharged amino acid, or a combination thereof; and wherein attachment ofsaid charged functional group to said nucleobase results in lesseraggregation of a plurality of bio-probes, as compared to bio-probes notcomprising a charged functional group attached to said nucleobase.2-116. (canceled)